Mapping of a N-terminal -helix domain required for human PINK1 stabilisation, Serine228 autophosphorylation and activation in cells

Kakade Poonam; Ojha Hina; Raimi Olawale G.; Shaw Andrew; Waddell Andrew D.; Ault James R; Burel Sophie; Brockmann Kathrin; Kumar Atul; Ahangar Mohd Syed; Krysztofinska Ewelina M.; Macartney Thomas; Bayliss Richard; Fitzgerald Julia C.; Muqit Miratul M. K.

doi:10.5281/zenodo.5767328

Published December 15, 2021 | Version v1

Journal article Open

Mapping of a N-terminal -helix domain required for human PINK1 stabilisation, Serine228 autophosphorylation and activation in cells

Human autosomal recessive mutations in the PINK1 gene are causal for Parkinson’s disease (PD). PINK1 encodes a mitochondrial localised protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function, however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain are unknown. We have employed mutagenesis studies of human PINK1 in cells to define the minimal region of PINK1, required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Bioinformatic analysis of the region spanning Ile111 to the kinase domain and inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal a-helical domain extension (NTE domain) within this region corroborated by hydrogen/deuterium exchange mass spectrometry (HDX-MS) of recombinant insect PINK1 protein. The AlphaFold structure also predicts the NTE domain forms an intramolecular interaction with the C-terminal extension (CTE). Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE:CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228); and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE domain mutants do not affect intrinsic catalytic kinase activity but do disrupt PINK1 stabilisation at the mitochondrial Translocase of outer membrane (TOM) complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE:CTE interface towards PINK1 stabilisation and activation and show that loss of NTE:CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.

Description of files

Figure 1B

PINK1
- First 16 lanes. Other lanes are from unrelated experiments
- Lane 1: Marker, Lane 2-3: hPINK1 WT (-CCCP), Lane 4-5: hPINK1 WT (+CCCP 3 hour), Lane 6-7: hPINK1 KI (-CCCP), Lane 8-9: hPINK1 KI (+CCCP 3 hour), Lane 10-11: OPA3₁₁₁hPINK1 (-CCCP), Lane 12-13: OPA3₁₁₁hPINK1 (+CCCP 3 hour), Lane 14-15: HSP60₁₁₁hPINK1 (-CCCP), Lane 16-17: HSP60₁₁₁hPINK1 (+CCCP 3 hour)
OPA1 (High exposure)
- Lane 1: Marker, Lane 2-3: hPINK1 WT (-CCCP), Lane 4-5: hPINK1 WT (+CCCP 3 hour), Lane 6-7: hPINK1 KI (-CCCP), Lane 8-9: hPINK1 KI (+CCCP 3 hour), Lane 10-11: OPA3₁₁₁hPINK1 (-CCCP), Lane 12-13: OPA3₁₁₁hPINK1 (+CCCP 3 hour), Lane 14-15: HSP60₁₁₁hPINK1 (-CCCP), Lane 16-17: HSP60₁₁₁hPINK1 (+CCCP 3 hour)
GAPDH (Low exposure)
- Lane 1: Marker, Lane 2-3: hPINK1 WT (-CCCP), Lane 4-5: hPINK1 WT (+CCCP 3 hour), Lane 6-7: hPINK1 KI (-CCCP), Lane 8-9: hPINK1 KI (+CCCP 3 hour), Lane 10-11: OPA3₁₁₁hPINK1 (-CCCP), Lane 12-13: OPA3₁₁₁hPINK1 (+CCCP 3 hour), Lane 14-15: HSP60₁₁₁hPINK1 (-CCCP), Lane 16-17: HSP60₁₁₁hPINK1 (+CCCP 3 hour)

Figure 1C

Figure 1C OPA1:
- Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA₁₁₁hPINK1 (-CCCP), Lane 16-17: OPA₁₁₁hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60₁₁₁hPINK1 (-CCCP), Lane 20-21: HSP60₁₁₁hPINK1 (+CCCP 3 hour)
Figure 1C Parkin:
- Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA₁₁₁hPINK1 (-CCCP), Lane 16-17: OPA₁₁₁hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60₁₁₁hPINK1 (-CCCP), Lane 20-21: HSP60₁₁₁hPINK1 (+CCCP 3 hour)
Figure 1C PINK1:
- Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA₁₁₁hPINK1 (-CCCP), Lane 16-17: OPA₁₁₁hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60₁₁₁hPINK1 (-CCCP), Lane 20-21: HSP60₁₁₁hPINK1 (+CCCP 3 hour)
Figure 1C pS65 Parkin:
- Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA₁₁₁hPINK1 (-CCCP), Lane 16-17: OPA₁₁₁hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60₁₁₁hPINK1 (-CCCP), Lane 20-21: HSP60₁₁₁hPINK1 (+CCCP 3 hour)
Figure 1C pUb:
- Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA₁₁₁hPINK1 (-CCCP), Lane 16-17: OPA₁₁₁hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60₁₁₁hPINK1 (-CCCP), Lane 20-21: HSP60₁₁₁hPINK1 (+CCCP 3 hour)
Figure 1C Vinculin:
- Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 3 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 3 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 3 hour), Lane 14-15: OPA₁₁₁hPINK1 (-CCCP), Lane 16-17: OPA₁₁₁hPINK1 (+CCCP 3 hour), Lane 18-19: HSP60₁₁₁hPINK1 (-CCCP), Lane 20-21: HSP60₁₁₁hPINK1 (+CCCP 3 hour)

Figure 1D

Figure 1D OPA1, PINK1 and GAPDH:
- Upper left: blot 1:
  - Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O) (not shown in paper), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) (not shown in paper), Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
- Lower left blot 2:
  - Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: PINK1(1-34)-PINK1(111- 581), (-A/O), Lane 16-17: PINK1(1-34)-PINK1(111- 581), (+A/O 9 hour).
- Upper right blot 3:
  - Lane 1: Marker, Lane 2-3: pOTC(1-30)-PINK1(111-581) (-A/O), Lane 4-5: pOTC(1-30)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (-A/O), Lane 8-9: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), Lane 10: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), (not shown in paper), Lane 11-12: pF1ATPase(1-30)- PINK1(111-581) (-A/O), Lane 13-14: pF1ATPase(1-30)- PINK1(111-581) (+A/O 9 hour) lane 15-16: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (-A/O), lane 17-18: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (+A/O 9 hour).
- Lower right: blot 4: (not shown in paper)
  - Lane 1: Marker, Lane 2-3: pALDH(1-19)-PINK1(111-581) (-A/O), Lane 4-5: pALDH(1-19)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pALDH (1-19) -(L15A) PINK1(111-581) (-A/O), Lane 8-9: pALDH (1-19) -(L15A) PINK1(111-581) (+A/O 9 hour.

Figure 1D pUb:

Upper left: blot 1:
- Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O) (not shown in paper), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) (not shown in paper), Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
Lower left blot 2:
- Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: PINK1(1-34)-PINK1(111- 581), (-A/O), Lane 16-17: PINK1(1-34)-PINK1(111- 581), (+A/O 9 hour).
Upper right blot 3:
- Lane 1: Marker, Lane 2-3: pOTC(1-30)-PINK1(111-581) (-A/O), Lane 4-5: pOTC(1-30)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (-A/O), Lane 8-9: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), Lane 10: pOTC(1-30)-(L5A/L8A/L9A) PINK1(111-581) (+A/O 9 hour), (not shown in paper), Lane 11-12: pF1ATPase(1-30)- PINK1(111-581) (-A/O), Lane 13-14: pF1ATPase(1-30)- PINK1(111-581) (+A/O 9 hour) lane 15-16: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (-A/O), lane 17-18: pF1- b ATPase (W29A/C32A/M33A) (1-30)- PINK1(111-581) (+A/O 9 hour).
Lower right: blot 4: (not shown in paper)
- Lane 1: Marker, Lane 2-3: pALDH(1-19)-PINK1(111-581) (-A/O), Lane 4-5: pALDH(1-19)-PINK1(111-581) (+A/O 9 hour), Lane 6-7: pALDH (1-19) -(L15A) PINK1(111-581) (-A/O), Lane 8-9: pALDH(1-19)-(L15A) PINK1(111-581) (+A/O 9 hour).

Figure 2B

Figure 2B pUb:
- Upper left: blot 1:
  - Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
- Upper right blot 2:
  - Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour).
- Lower left blot 3:
  - Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour).

Figure 2B PINK1:
- Upper left: blot 1:
  - Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
- Upper right blot 2:
  - Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour).
- Lower left blot 3:
  - Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour).

Figure 2B OPA1:
- Upper left: blot 1:
  - Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
- Upper right blot 2:
  - Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour).
- Lower left blot 3:
  - Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour).

Figure 2B GAPDH:
- Upper left: blot 1:
  - Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
- Upper right blot 2:
  - Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(115- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(115- 581), (+A/O 9 hour).
- Lower left blot 3:
  - Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(120- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(120- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(125- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(125- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(130- 581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(130- 581) (+A/O 9 hour).

Figure 2C

Figure 2C pUb:
- Blot 1:
  - Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
- Blot 2:
  - Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(112- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(112- 581), (+A/O 9 hour).
- Blot 3:
  - Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(113- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(113- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(114- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(114- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(115-581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(115- 581) (+A/O 9 hour).

Figure 2C PINK1:
- Blot 1:
  - Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
- Blot 2:
  - Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(112- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(112- 581), (+A/O 9 hour).
- Blot 3:
  - Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(113- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(113- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(114- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(114- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(115-581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(115- 581) (+A/O 9 hour).

Figure 2C OPA1:
- Blot 1:
  - Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
- Blot 2:
  - Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(112- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(112- 581), (+A/O 9 hour).
- Blot 3:
  - Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(113- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(113- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(114- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(114- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(115-581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(115- 581) (+A/O 9 hour).

Figure 2C GAPDH:
- Blot 1:
  - Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
- Blot 2:
  - Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60 (1-27)-PINK1(112- 581), (-A/O), Lane 16-17: HSP60 (1-27)-PINK1(112- 581), (+A/O 9 hour).
- Blot 3:
  - Lane 1: Marker, Lane 2-3: HSP60 (1-27)-PINK1(113- 581) (-A/O), Lane 4-5: HSP60 (1-27)-PINK1(113- 581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(114- 581) (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(114- 581) (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(115-581) (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(115- 581) (+A/O 9 hour).

Figure 2D

Figure 2D PINK1, OPA1 and GAPDH:
- Upper left Blot 1:
  - Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O) (not shown in paper), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) (not shown in paper), Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
- Lower left Blot 2:
  - Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60(1-27)-PINK1(111-581)-(E112K) (-A/O), Lane 16-17: HSP60(1-27)-PINK1(111-581)-(E112K) (+A/O 9 hour).
- Upper Right Blot 3:
  - Lane 1: Marker, Lane 2-3: HSP60(1-27)-PINK1(111-581)-(E113K) (-A/O), Lane 4-5: HSP60(1-27)-PINK1(111-581)-(E113K) (+A/O 9 hour), Lane 6-7: HSP60(1-27)-PINK1(111-581)-(E117K) (-A/O), Lane 8-9: HSP60(1-27)-PINK1(111-581)-(E117K) (+A/O 9 hour), Lane 10-11: HSP60(1-27)-PINK1(111-581)-(E112+E113K) (-A/O), Lane 12-13: HSP60(1-27)-PINK1(111-581)-(E112K+E113K) (+A/O 9 hour), Lane 14-15: HSP60(1-27)-PINK1(111-581)-(E112+E113K+E117K) (-A/O), Lane 16-17: HSP60(1-27)-PINK1(111-581)-(E112+E113K+E117K) (+A/O 9 hour).
- Lower Right Blot 4: (Blot is from unrelated experiment)

Figure 2D P-Ub:
- left Blot 1:
  - Lane 1: Marker, Lane 2-3: Human SKOV3 PINK1 WT (-A/O) (not shown in paper), Lane 4-5: Human SKOV3 PINK1 WT (+A/O 9 hour) (not shown in paper), Lane 6-7: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (-A/O), Lane 8-9: Human SKOV3 PINK1 knockout 3xFLAG alone (Veh) (+A/O 9 hour), Lane 10-11: hPINK1 WT (-A/O), Lane 12-13: hPINK1 WT (+A/O 9 hour), Lane 14-15: hPINK1 KI (D384A) (-A/O), Lane 16-17: hPINK1 KI (D384A) (+A/O 9 hour).
- left Blot 2:
  - Lane 1: Marker, Lane 2-3: ₁₁₁PINK1(111-581) (-A/O), Lane 4-5: ₁₁₁PINK1(111-581) (+A/O 9 hour), Lane 6-7: HSP60 (1-27)-PINK1(104-581), (-A/O), Lane 8-9: HSP60 (1-27)-PINK1(104-581), (+A/O 9 hour), Lane 10-11: HSP60 (1-27)-PINK1(111-581), (-A/O), Lane 12-13: HSP60 (1-27)-PINK1(111-581), (+A/O 9 hour), Lane 14-15: HSP60(1-27)-PINK1(111-581)-(E112K) (-A/O), Lane 16-17: HSP60(1-27)-PINK1(111-581)-(E112K) (+A/O 9 hour).
- Right Blot 3:
  - Lane 1: Marker, Lane 2-3: HSP60(1-27)-PINK1(111-581)-(E113K) (-A/O), Lane 4-5: HSP60(1-27)-PINK1(111-581)-(E113K) (+A/O 9 hour), Lane 6-7: HSP60(1-27)-PINK1(111-581)-(E117K) (-A/O), Lane 8-9: HSP60(1-27)-PINK1(111-581)-(E117K) (+A/O 9 hour), Lane 10-11: HSP60(1-27)-PINK1(111-581)-(E112+E113K) (-A/O), Lane 12-13: HSP60(1-27)-PINK1(111-581)-(E112K+E113K) (+A/O 9 hour), Lane 14-15: HSP60(1-27)-PINK1(111-581)-(E112+E113K+E117K) (-A/O), Lane 16-17: HSP60(1-27)-PINK1(111-581)-(E112+E113K+E117K) (+A/O 9 hour).

Right Blot 4: (Blot is from unrelated experiment)

Figure 4A

Figure 4A HSP60:
- Upper blot:
  - Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG empty (-O/A), Lane 5-6: FLAG empty (+O/A), Lane 7-8: hPINK1 WT (-O/A), Lane 9-10: hPINK1 WT (+O/A), Lane 11-12: hPINK1 KI (-O/A), Lane 13-14: hPINK1 KI (+O/A), Lane 15-16: C125G(-O/A), Lane 17-18: C125G (+O/A), Lane 19-20: Q126P (-O/A), Lane 21-22: Q126P (+O/A), Lane 23-24: A168P(-O/A), Lane 25-26: A168P (+O/A)
- Lower blot:
  - Lane 1: Marker, Lane 2: empty, Lane 3-4: E240K (-O/A), Lane 5-6: E240K (+O/A), Lane 7-8: G309D (-O/A), Lane 9-10: G309D (+O/A), Lane 11-12: G409V (-O/A), Lane 13-14: G409V (+O/A), Lane 15-16: L539F (-O/A), Lane 17-18: L539F (+O/A), Lane 19-20: 534_535insQ (-O/A), Lane 21-22: 534_535insQ (+O/A)
Figure 4A OPA1:
- Upper blot:
  - Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG empty (-O/A), Lane 5-6: FLAG empty (+O/A), Lane 7-8: hPINK1 WT (-O/A), Lane 9-10: hPINK1 WT (+O/A), Lane 11-12: hPINK1 KI (-O/A), Lane 13-14: hPINK1 KI (+O/A), Lane 15-16: C125G(-O/A), Lane 17-18: C125G (+O/A), Lane 19-20: Q126P (-O/A), Lane 21-22: Q126P (+O/A), Lane 23-24: A168P(-O/A), Lane 25-26: A168P (+O/A)
- Lower blot:
  - Lane 1: Marker, Lane 2: empty, Lane 3-4: E240K (-O/A), Lane 5-6: E240K (+O/A), Lane 7-8: G309D (-O/A), Lane 9-10: G309D (+O/A), Lane 11-12: G409V (-O/A), Lane 13-14: G409V (+O/A), Lane 15-16: L539F (-O/A), Lane 17-18: L539F (+O/A), Lane 19-20: 534_535insQ (-O/A), Lane 21-22: 534_535insQ (+O/A)
Figure 4A PINK1:
- Upper blot:
  - Lane 1: Marker, Lane 2: empty, Lane 3-4: E240K (-O/A), Lane 5-6: E240K (+O/A), Lane 7-8: G309D (-O/A), Lane 9-10: G309D (+O/A), Lane 11-12: G409V (-O/A), Lane 13-14: G409V (+O/A), Lane 15-16: L539F (-O/A), Lane 17-18: L539F (+O/A), Lane 19-20: 534_535insQ (-O/A), Lane 21-22: 534_535insQ (+O/A)
- Lower blot:
  - Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG empty (-O/A), Lane 5-6: FLAG empty (+O/A), Lane 7-8: hPINK1 WT (-O/A), Lane 9-10: hPINK1 WT (+O/A), Lane 11-12: hPINK1 KI (-O/A), Lane 13-14: hPINK1 KI (+O/A), Lane 15-16: C125G(-O/A), Lane 17-18: C125G (+O/A), Lane 19-20: Q126P (-O/A), Lane 21-22: Q126P (+O/A), Lane 23-24: A168P(-O/A), Lane 25-26: A168P (+O/A)

Figure 4A pS228A PINK1:
- Upper blot:
  - Lane 1: Marker, Lane 2: empty, Lane 3-4: E240K (-O/A), Lane 5-6: E240K (+O/A), Lane 7-8: G309D (-O/A), Lane 9-10: G309D (+O/A), Lane 11-12: G409V (-O/A), Lane 13-14: G409V (+O/A), Lane 15-16: L539F (-O/A), Lane 17-18: L539F (+O/A), Lane 19-20: 534_535insQ (-O/A), Lane 21-22: 534_535insQ (+O/A)
- Lower blot:
  - Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG empty (-O/A), Lane 5-6: FLAG empty (+O/A), Lane 7-8: hPINK1 WT (-O/A), Lane 9-10: hPINK1 WT (+O/A), Lane 11-12: hPINK1 KI (-O/A), Lane 13-14: hPINK1 KI (+O/A), Lane 15-16: C125G(-O/A), Lane 17-18: C125G (+O/A), Lane 19-20: Q126P (-O/A), Lane 21-22: Q126P (+O/A), Lane 23-24: A168P(-O/A), Lane 25-26: A168P (+O/A)

Figure 4A pS65Ub:
- Upper blot:
  - Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG empty (-O/A), Lane 5-6: FLAG empty (+O/A), Lane 7-8: hPINK1 WT (-O/A), Lane 9-10: hPINK1 WT (+O/A), Lane 11-12: hPINK1 KI (-O/A), Lane 13-14: hPINK1 KI (+O/A), Lane 15-16: C125G(-O/A), Lane 17-18: C125G (+O/A), Lane 19-20: Q126P (-O/A), Lane 21-22: Q126P (+O/A), Lane 23-24: A168P(-O/A), Lane 25-26: A168P (+O/A)
- Lower blot:
  - Lane 1: Marker, Lane 2: empty, Lane 3-4: E240K (-O/A), Lane 5-6: E240K (+O/A), Lane 7-8: G309D (-O/A), Lane 9-10: G309D (+O/A), Lane 11-12: G409V (-O/A), Lane 13-14: G409V (+O/A), Lane 15-16: L539F (-O/A), Lane 17-18: L539F (+O/A), Lane 19-20: 534_535insQ (-O/A), Lane 21-22: 534_535insQ (+O/A)

Figure 4D

Figure 4D OPA1
- Top membrane first 12 lanes
  - Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 6 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 6 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 6 hour)
- Middle membrane: from unrelated experiment
- Bottom membrane first 16 lanes
  - Lane 1: Marker, Lane 2-3: V528A (-CCCP), Lane 4-5: V528A (+CCCP 6 hour), Lane 6-7: L532A (-CCCP), Lane 8-9: L532A (+CCCP 6 hour), Lane 10-11: L539A (-CCCP), Lane 12-13: L539A (+CCCP 6 hour), Lane 14-15: 3A (-CCCP), Lane 16-17: FLAG-emp (+CCCP 6 hour)
Figure 4D Total 8a
- Bottom membrane first 12 lanes (Other lanes are from unrelated experiments)
  - Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 6 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 6 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 6 hour)
- Top membrane first 16 lanes (Other lanes are from unrelated experiments)
  - Lane 1: Marker, Lane 2-3: V528A (-CCCP), Lane 4-5: V528A (+CCCP 6 hour), Lane 6-7: L532A (-CCCP), Lane 8-9: L532A (+CCCP 6 hour), Lane 10-11: L539A (-CCCP), Lane 12-13: L539A (+CCCP 6 hour), Lane 14-15: 3A (-CCCP), Lane 16-17: FLAG-emp (+CCCP 6 hour)
Figure 4D pS111 Rab8a
- Top membrane all lanes
  - Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 6 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 6 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 6 hour)
- 2^nd membrane: From unrelated experiment
- 3^rd membrane all lanes.
  - Lane 1: Marker, Lane 2-3: V528A (-CCCP), Lane 4-5: V528A (+CCCP 6 hour), Lane 6-7: L532A (-CCCP), Lane 8-9: L532A (+CCCP 6 hour), Lane 10-11: L539A (-CCCP), Lane 12-13: L539A (+CCCP 6 hour), Lane 14-15: 3A (-CCCP), Lane 16-17: FLAG-emp (+CCCP 6 hour)
- 4^th membrane: From unrelated experiment
Figure 4D HSP60
- Top membrane
  - Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 6 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 6 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 6 hour)
- Middle membrane: from unrelated experiment
- Bottom membrane
  - Lane 1: Marker, Lane 2-3: V528A (-CCCP), Lane 4-5: V528A (+CCCP 6 hour), Lane 6-7: L532A (-CCCP), Lane 8-9: L532A (+CCCP 6 hour), Lane 10-11: L539A (-CCCP), Lane 12-13: L539A (+CCCP 6 hour), Lane 14-15: 3A (-CCCP), Lane 16-17: FLAG-emp (+CCCP 6 hour)
Figure 4D pS65UB
- Top membrane first 12 lanes (Other lanes are from unrelated experiments)
  - Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 6 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 6 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 6 hour)
- Bottom membrane first 16 lanes (Other lanes are from unrelated experiments)
  - Lane 1: Marker, Lane 2-3: V528A (-CCCP), Lane 4-5: V528A (+CCCP 6 hour), Lane 6-7: L532A (-CCCP), Lane 8-9: L532A (+CCCP 6 hour), Lane 10-11: L539A (-CCCP), Lane 12-13: L539A (+CCCP 6 hour), Lane 14-15: 3A (-CCCP), Lane 16-17: FLAG-emp (+CCCP 6 hour)
Figure 4D PINK1
- Top membrane
  - Lane 1: Marker, Lane 2-3: FLAG-emp (-CCCP), Lane 4-5: FLAG-emp (+CCCP 6 hour), Lane 6-7: hPINK1 WT (-CCCP), Lane 8-9: hPINK1 WT (+CCCP 6 hour), Lane 10-11: hPINK1 KI (-CCCP), Lane 12-13: hPINK1 KI (+CCCP 6 hour)
- Middle membrane: from unrelated experiment
- Bottom membrane
  - Lane 1: Marker, Lane 2-3: V528A (-CCCP), Lane 4-5: V528A (+CCCP 6 hour), Lane 6-7: L532A (-CCCP), Lane 8-9: L532A (+CCCP 6 hour), Lane 10-11: L539A (-CCCP), Lane 12-13: L539A (+CCCP 6 hour), Lane 14-15: 3A (-CCCP), Lane 16-17: FLAG-emp (+CCCP 6 hour)

Figure 4E

Figure 4E pS65Ub
- First panel lanes 1-13
  - Lane 1: Marker, Lane 2-4: hPINK1 WT (-CCCP), Lane 5-7: hPINK1WT (+CCCP 3 hour), Lane 8-10: hPINK1 KI (-CCCP), Lane 11-13: hPINK1 KI (+ CCCP 3 hour)
- Second panel wells 8-13 (Lanes 1-7 not included in paper)
  - Lane 1: Marker, Lane 8-10: 3A (-CCCP), Lane 11-13: 3A (+CCCP 3 hour)
Figure 4E Ubiqutin
- First panel lanes 1-13
  - Lane 1: Marker, Lane 2-4: hPINK1 WT (-CCCP), Lane 5-7: hPINK1WT (+CCCP 3 hour), Lane 8-10: hPINK1 KI (-CCCP), Lane 11-13: hPINK1 KI (+ CCCP 3 hour)
- Second panel wells 8-13 (Lanes 1-7 not included in paper)
  - Lane 1: Marker, Lane 8-10: 3A (-CCCP), Lane 11-13: 3A (+CCCP 3 hour)
Figure 4E PINK1
Upper panel of blot
- Lanes 1-13 and Lanes 20-25 (Lanes 14-19 not included in paper)
  - Lane 1: Marker, Lane 2-4: hPINK1 WT (-CCCP), Lane 5-7: hPINK1WT (+CCCP 3 hour), Lane 8-10: hPINK1 KI (-CCCP), Lane 11-13: hPINK1 KI (+ CCCP 3 hour), Lane 20-22: 3A (-CCCP), Lane 23-25: 3A (+CCCP 3 hour)
Figure 4E Coomassie
- Lanes 1-13 and Lanes 20-25 (Lanes 14-19 not included in paper)
  - Lane 1: Marker, Lane 2-4: hPINK1 WT (-CCCP), Lane 5-7: hPINK1WT (+CCCP 3 hour), Lane 8-10: hPINK1 KI (-CCCP), Lane 11-13: hPINK1 KI (+ CCCP 3 hour), Lane 20-22: 3A (-CCCP), Lane 23-25: 3A (+CCCP 3 hour)

Figure 5

Figure 5 GAPDH:
- Lane 1: Marker, Lane 2-3: Control (- CCCP), Lane 4-5: Control (3 hour CCCP), Lane 6-7: Control (6 hour CCCP), Lane 8-9: hPINK1 mutation Q126P (- CCCP), Lane 10-11: hPINK1 mutation Q126P (3 hour CCCP), Lane 12-13: hPINK1 mutation Q126P (6 hour CCCP)
Figure 5 OPA1:
- Lane 1: Marker, Lane 2-3: Control (- CCCP), Lane 4-5: Control (3 hour CCCP), Lane 6-7: Control (6 hour CCCP), Lane 8-9: hPINK1 mutation Q126P (- CCCP), Lane 10-11: hPINK1 mutation Q126P (3 hour CCCP), Lane 12-13: hPINK1 mutation Q126P (6 hour CCCP)
Figure 5 Rab8A:
- Lane 1: Marker, Lane 2-3: Control (- CCCP), Lane 4-5: Control (3 hour CCCP), Lane 6-7: Control (6 hour CCCP), Lane 8-9: hPINK1 mutation Q126P (- CCCP), Lane 10-11: hPINK1 mutation Q126P (3 hour CCCP), Lane 12-13: hPINK1 mutation Q126P (6 hour CCCP)
Figure 5 Rab8A pS111:
- Lane 1: Marker, Lane 2-3: Control (- CCCP), Lane 4-5: Control (3 hour CCCP), Lane 6-7: Control (6 hour CCCP), Lane 8-9: hPINK1 mutation Q126P (- CCCP), Lane 10-11: hPINK1 mutation Q126P (3 hour CCCP), Lane 12-13: hPINK1 mutation Q126P (6 hour CCCP)
Figure 5 pS65Ub TUBE pull-down:
- Lane 1: Marker, Lane 2-3: Control (- CCCP), Lane 4-5: Control (3 hour CCCP), Lane 6-7: Control (6 hour CCCP), Lane 8-9: hPINK1 mutation Q126P (- CCCP), Lane 10-11: hPINK1 mutation Q126P (3 hour CCCP), Lane 12-13: hPINK1 mutation Q126P (6 hour CCCP), Lane 14: HEK-293 PINK1 KO (3 hour CCCP), Lane 15: HEK-293 PINK1 KO + PINK1-3FLAG (3 hour CCCP)
Figure 5 PINK1 IP:
- Lane 1: Marker, Lane 2-3: Control (- CCCP), Lane 4-5: Control (3 hour CCCP), Lane 6-7: Control (6 hour CCCP), Lane 8-9: hPINK1 mutation Q126P (- CCCP), Lane 10-11: hPINK1 mutation Q126P (3 hour CCCP), Lane 12-13: hPINK1 mutation Q126P (6 hour CCCP), Lane 14: HEK-293 PINK1 KO (3 hour CCCP), Lane 15: HEK-293 PINK1 KO + PINK1-3FLAG (3 hour CCCP)

Supplementary Figure 1

Supplementary Figure 1 PINK High
- Lane1: Marker, Lane 2-3: HEK 293 (-CCCP), Lane 4-5: HEK 293 (CCCP 3 hour), Lane 6-7: HEK 293 PINK1 KO (- CCCP), Lane 8-9: HEK 293 PINK1 KO (CCCP 3 hour), Lane 10-11: HEK 293 PINK1 KO + hPINK1-3FLAG (- CCCP), Lane 12-13: HEK 293 PINK1 KO + hPINK1-3FLAG (CCCP 3 hour)
Supplementary Figure 1 PINK low
- Lane1: Marker, Lane 2-3: HEK 293 (-CCCP), Lane 4-5: HEK 293 (CCCP 3 hour), Lane 6-7: HEK 293 PINK1 KO (- CCCP), Lane 8-9: HEK 293 PINK1 KO (CCCP 3 hour), Lane 10-11: HEK 293 PINK1 KO + hPINK1-3FLAG (- CCCP), Lane 12-13: HEK 293 PINK1 KO + hPINK1-3FLAG (CCCP 3 hour)
Supplementary Figure 1 PINK pS65 Ub and HSP60
- Upper gel: HSP60, Lower gel: ps65 Ub
- Lane1: Marker Lane 2: empty, Lane 3-4: HEK 293 (-CCCP), Lane 5-6: HEK 293 (CCCP 3 hour), Lane 7-8: HEK 293 PINK1 KO (- CCCP), Lane 9-10: HEK 293 PINK1 KO (CCCP 3 hour), Lane 11-12: HEK 293 PINK1 KO + hPINK1-3FLAG (- CCCP), Lane 13-14: HEK 293 PINK1 KO + hPINK1-3FLAG (CCCP 3 hour)

Supplementary Figure 5

Lane 1 : marker; Lane 2: WT; Lane3: L507A, lane 4: L514F and lane6: I503A.

Supplementary Figure 7

Lane 1 : marker; Lane 2: L507A; Lane3: L514A, lane 4: I503A; lane6: WT; lane7: W129P and lane8: E125K

Supplementary Figure 8B

Supplementary Figure 8 B: PINK1
- Lane 1: Marker, Lane 2-3: Flp-In T-REx HeLa PINK1 KO (-A/O), Lane 4-5: Flp-In T-REx HeLa PINK1 KO (+A/O 9 hour) Lane 6-7: Flp-In T-REx HeLa PINK1 KO transfected with GFP-hPINK1-104-END (-A/O) (Data not shown), Lane 8-9: Flp-In T-REx HeLa PINK1 KO transfected with GFP-hPINK1 104-END (+A/O 9 hour) (Data not shown), Lane 10-11: Flp-In T-REx HeLa PINK1 KO transfected with hPINK1 WT GFP (-A/O), Lane 12-13: Flp-In T-REx HeLa PINK1 KO transfected with hPINK1 WT GFP (+A/O 9 hour).

Supplementary Figure 8 B: OPA1
- Lane 1: Marker, Lane 2-3: Flp-In T-REx HeLa PINK1 KO (-A/O), Lane 4-5: Flp-In T-REx HeLa PINK1 KO (+A/O 9 hour) Lane 6-7: Flp-In T-REx HeLa PINK1 KO transfected with GFP-hPINK1-104-END (-A/O) (Data not shown), Lane 8-9: Flp-In T-REx HeLa PINK1 KO transfected with GFP-hPINK1 104-END (+A/O 9 hour) (Data not shown), Lane 10-11: Flp-In T-REx HeLa PINK1 KO transfected with hPINK1 WT GFP (-A/O), Lane 12-13: Flp-In T-REx HeLa PINK1 KO transfected with hPINK1 WT GFP (+A/O 9 hour).

Supplementary Figure 8 B: GAPDH
- Lane 1: Marker, Lane 2-3: Flp-In T-REx HeLa PINK1 KO (-A/O), Lane 4-5: Flp-In T-REx HeLa PINK1 KO (+A/O 9 hour) Lane 6-7: Flp-In T-REx HeLa PINK1 KO transfected with GFP-hPINK1-104-END (-A/O) (Data not shown), Lane 8-9: Flp-In T-REx HeLa PINK1 KO transfected with GFP-hPINK1 104-END (+A/O 9 hour) (Data not shown), Lane 10-11: Flp-In T-REx HeLa PINK1 KO transfected with hPINK1 WT GFP (-A/O), Lane 12-13: Flp-In T-REx HeLa PINK1 KO transfected with hPINK1 WT GFP (+A/O 9 hour).
Supplementary Figure 8 B: PSer65 Ubiquitin
- Lane 1: Marker, Lane 2-3: Flp-In T-REx HeLa PINK1 KO (-A/O), Lane 4-5: Flp-In T-REx HeLa PINK1 KO (+A/O 9 hour) Lane 6-7: Flp-In T-REx HeLa PINK1 KO transfected with GFP-hPINK1-104-END (-A/O) (Data not shown), Lane 8-9: Flp-In T-REx HeLa PINK1 KO transfected with GFP-hPINK1 104-END (+A/O 9 hour) (Data not shown), Lane 10-11: Flp-In T-REx HeLa PINK1 KO transfected with hPINK1 WT GFP (-A/O), Lane 12-13: Flp-In T-REx HeLa PINK1 KO transfected with hPINK1 WT GFP (+A/O 9 hour).

Supplementary Figure 9A

Supplementary figure 9A pS65Ub
- Upper blot
  - Lanes 1-13 and Lanes 17-19 (Lanes 14-16 not included in paper)
  - Lane 1: Marker, Lane 2-4: hPINK1 WT (-CCCP), Lane 5-7: hPINK1WT (+CCCP 3 hour), Lane 8-10: hPINK1 KI (-CCCP), Lane 11-13: hPINK1 KI (+ CCCP 3 hour), Lane 17-19: hPINK WT (+CCCP 3 hour)
Supplementary figure 9A Ub
- Lower blot
  - Lanes 1-13 and Lanes 17-19 (Lanes 14-16 not included in paper)
  - Lane 1: Marker, Lane 2-4: hPINK1 WT (-CCCP), Lane 5-7: hPINK1WT (+CCCP 3 hour), Lane 8-10: hPINK1 KI (-CCCP), Lane 11-13: hPINK1 KI (+ CCCP 3 hour), Lane 17-19: hPINK WT (+CCCP 3 hour)
Supplementary figure 9A PINK1
- Lower blot
  - Lanes 1-13 and Lanes 17-19 (Lanes 14-16 not included in paper)
  - Lane 1: Marker, Lane 2-4: hPINK1 WT (-CCCP), Lane 5-7: hPINK1WT (+CCCP 3 hour), Lane 8-10: hPINK1 KI (-CCCP), Lane 11-13: hPINK1 KI (+ CCCP 3 hour), Lane 17-19: hPINK WT (+CCCP 3 hour)
Supplementary figure 9A Commassie
- Lanes 1-13 and Lanes 17-19 (Lanes 14-16 not included in paper)
- Lane 1: Marker, Lane 2-4: hPINK1 WT (-CCCP), Lane 5-7: hPINK1WT (+CCCP 3 hour), Lane 8-10: hPINK1 KI (-CCCP), Lane 11-13: hPINK1 KI (+ CCCP 3 hour), Lane 17-19: hPINK WT (+CCCP 3 hour)

Supplementary Figure 9B

Supplementary Figure 9B PINK1
- Lower Panel
  - Lane 1: Marker, Other lanes with increasing time point of hPINK1 WT
Supplementary Figure 9B Ubiquitin
- Right side panel: Upper and lower blot: increasing time points of hPINK1 WT

Supplementary Figure 9B pUbiquitin
- Upper and lower panel: increasing time points of hPINK1 WT
Supplementary Figure 9B Coomassie
- Lane 1: Marker, Other lanes with increasing time point of hPINK1 WT

Supplementary Figure 10A

Supplementary Figure 10A Coomassie
- Lane 1: Marker, Lane 2: empty, Lane 3-4: PhCPINK1+TetraUb, Lane 5-6: PhCPINK1 KI+TetraUb, Lane 7-8: E125K+TetraUb. Lane 9-10: C128G+TetraUb, Lane 11-12: W129P+TetraUb, Lane 13-14: I503A+TetraUb, Lane 15-16: L507A+TetraUb, Lane 17-18: L514F+TetraUb
Supplementary Figure 10A Autoradiograph
- Lane 1: Marker, Lane 2: empty, Lane 3-4: PhCPINK1+TetraUb, Lane 5-6: PhCPINK1 KI+TetraUb, Lane 7-8: E125K+TetraUb. Lane 9-10: C128G+TetraUb, Lane 11-12: W129P+TetraUb, Lane 13-14: I503A+TetraUb, Lane 15-16: L507A+TetraUb, Lane 17-18: L514F+TetraUb

Supplementary Figure 10B

Supplementary Figure 10B BN-PAGE
- Lane 1: Marker, Lane 2: hPINK1 WT (- A/O), Lane 3: hPINK1 WT (+A/O), Lane 4: FLAG-empty (+A/O), Lane 5: hPINK1 KI (+A/O), Lane 6: S228A (+A/O), Lane 7: C125G (+A/O), Lane 8: Q126P (+A/O), Lane 9: ins534Q_535Q (+A/O)
Supplementary Figure 10B HSP60
- Lane 1: Marker, Lane 2: hPINK1 WT (- A/O), Lane 3: hPINK1 WT (+A/O), Lane 4: FLAG-empty (+A/O), Lane 5: hPINK1 KI (+A/O), Lane 6: S228A (+A/O), Lane 7: C125G (+A/O), Lane 8: Q126P (+A/O), Lane 9: ins534Q_535Q (+A/O)
Supplementary Figure 10B OPA
- Lane 1: Marker, Lane 2: empty, Lane 3: hPINK1 WT (- A/O), Lane 4: hPINK1 WT (+A/O), Lane 5: FLAG-empty (+A/O), Lane 6: hPINK1 KI (+A/O), Lane 7: S228A (+A/O), Lane 8: C125G (+A/O), Lane 9: Q126P (+A/O), Lane 10: ins534Q_535Q (+A/O)
Supplementary Figure 10B DCP (PINK1)
- Lane 1: Marker, Lane 2: empty, Lane 3: hPINK1 WT (- A/O), Lane 4: hPINK1 WT (+A/O), Lane 5: FLAG-empty (+A/O), Lane 6: hPINK1 KI (+A/O), Lane 7: S228A (+A/O), Lane 8: C125G (+A/O), Lane 9: Q126P (+A/O), Lane 10: ins534Q_535Q (+A/O)
Supplementary Figure 10B pS65Ub
- Lane 1: Marker, Lane 2: empty, Lane 3: hPINK1 WT (- A/O), Lane 4: hPINK1 WT (+A/O), Lane 5: FLAG-empty (+A/O), Lane 6: hPINK1 KI (+A/O), Lane 7: S228A (+A/O), Lane 8: C125G (+A/O), Lane 9: Q126P (+A/O), Lane 10: ins534Q_535Q (+A/O)

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Figure_1B_GAPDH.tif

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Figure_1B_GAPDH.tif md5:5f0a43f5b896eae6de0b0d2acb78b97f	250.4 kB	Preview Download
Figure_1B_opa1.tif md5:0c7efe70ccc4701894ee7d86a2de3726	529.4 kB	Preview Download
Figure_1B_PINK1.tif md5:6d1519cc92f745126159f0f1fad44f1e	1.1 MB	Preview Download
Figure_1C_OPA1.tif md5:42df688e52e31a61db80387a11ac0fc8	951.1 kB	Preview Download
Figure_1C_Parkin.tif md5:bdabdab0b9a26ece321fb079481d4940	1.9 MB	Preview Download
Figure_1C_PINK1.tif md5:a9306f3c151289044e6c4c3970753a25	1.2 MB	Preview Download
Figure_1C_pS65_parkin.tif md5:4ef32b3f4060f472820e1d15ee9bbb00	1.9 MB	Preview Download
Figure_1C_pUb.tif md5:e783878b6815f121cdb158e7d9ad46a3	1.5 MB	Preview Download
Figure_1C_Vinculin.tif md5:39a63f45ed59cc1b9d31dee2aa30bb3f	893.1 kB	Preview Download
Figure_1D_OPA1 GAPDH and PINK1.tif md5:3a1c762a567144510dc45d3e2cf1e6dd	2.0 MB	Preview Download
Figure_1D_P-Ub.tif md5:0a6ee4e9a7bd8154b999287fa84209c4	2.3 MB	Preview Download
Figure_2B_GAPDH.tif md5:7f516a2d7bfb151bb0a84cd79cb558d5	785.1 kB	Preview Download
Figure_2B_OPA1.tif md5:4ee51086c989f7daa1b4f20ea5dfc81f	785.1 kB	Preview Download
Figure_2B_PINK1-DCP.tif md5:d157bde8f7a2ad21cb291be4a6cbe001	2.1 MB	Preview Download
Figure_2B_PS65Ub.tif md5:adab611e2d3dd6c171f3cb2b26e68927	1.3 MB	Preview Download
Figure_2C_GAPDH.tif md5:89cb9ca11378bcc311a6a9d364886bfa	805.4 kB	Preview Download
Figure_2C_OPA1.tif md5:959ca2f94a321ee40a476d8195d03097	877.3 kB	Preview Download
Figure_2C_P-Ub.tif md5:0d089affb26a7f42949638884be6693a	935.7 kB	Preview Download
Figure_2C_PINK1.tif md5:403830d88b02c700ff800d96d55b5b48	942.4 kB	Preview Download
Figure_2D_P-Ub1.tif md5:499635a77de7599c9eab7d4407a8298e	1.0 MB	Preview Download
Figure_2D_P-Ub2.tif md5:5ce0795be463aef50ea8a52d377cf159	1.1 MB	Preview Download
Figure_2D_PINK1 OPA1 and GAPDH.tif md5:b8d149fc4ead6a7c0227941ad7136105	2.2 MB	Preview Download
Figure_4A_HSP60.png md5:ce65c32f6801d2a50e3b515d1139edfd	114.0 kB	Preview Download
Figure_4A_OPA1.png md5:3a451111d3e4f0e86bff7c949612d2b0	286.7 kB	Preview Download
Figure_4A_PINK1.png md5:d71a112f6f3c5de7070fccc78751ede3	316.7 kB	Preview Download
Figure_4A_pS228A_PINK1.png md5:a863757b4fa92e00e1c1bab518f0ee27	453.7 kB	Preview Download
Figure_4A_pUb.png md5:330828ae5c53e0d92374a069340009d3	1.2 MB	Preview Download
Figure_4D_hsp60.tif md5:b4a37822ec35dc336f69425d0d49dd2f	2.7 MB	Preview Download
Figure_4D_opa1.tif md5:0e79737efd724403b33ba2eefaa17f3f	34.0 MB	Preview Download
Figure_4D_pink1.tif md5:2cd93d9b208111a8ae2b68a3436ecc84	1.1 MB	Preview Download
Figure_4D_pS111_Rab8a.tif md5:a1b4395539a793f83b6ab34a88892d43	32.3 MB	Preview Download
Figure_4D_pS65Ubb.tif md5:e0936cadde9eaa1c2383c6dbad7752ce	31.0 MB	Preview Download
Figure_4D_total_Rab8a.tif md5:4b130766c90a6e8bbf35e7d92f7ceb24	2.6 MB	Preview Download
Figure_4E_coomassie.tif md5:964ccdd3507b018c7573a6cacdc2ac1f	6.3 MB	Preview Download
Figure_4E_PINK1.tif md5:279a6bed5a758590c90cb9adafb8aefa	3.9 MB	Preview Download
Figure_4e_pS65Ub.tif md5:941f7968b3c4c0d45d7375224dce15fa	1.9 MB	Preview Download
Figure_4e_Ub.tif md5:a550824c45e50fd5c018f8a2cc122707	991.3 kB	Preview Download
Figure_5_GAPDH.tif md5:4b703f58cba653cfe8c48679c9e76c88	202.5 kB	Preview Download
Figure_5_OPA1.tif md5:12524fc0aca061c15c3b5139750f2707	394.1 kB	Preview Download
Figure_5_PINK1 Ip.tif md5:08b2cb3332d7c90b98342cd2732a30fd	10.6 MB	Preview Download
Figure_5_pS65Ub.tif md5:bae7b3324775d739602c1518c51f8a46	2.9 MB	Preview Download
Figure_5_Rab8A pS111.tif md5:a12d709c440311cd1df1bd42b4718cff	951.5 kB	Preview Download
Figure_5_Rab8A.tif md5:7bf846f772931b0442e9f8bff00af43d	224.7 kB	Preview Download
Supplementary Figure 8B GAPDH.tif md5:4cbbf3ea4443f066c9d2f4b715dd6045	5.2 MB	Preview Download
Supplementary Figure 8B opa1.tif md5:c77f4988aa96723285b7de3f39dbe903	5.2 MB	Preview Download
Supplementary Figure 8B PINK1.tif md5:e83a3827f62892da3a8b1236897c146f	8.4 MB	Preview Download
Supplementary Figure 8B ubiquitin.tif md5:c3ddc8e5ad7f3ed4688f5bc4007e4f2f	7.3 MB	Preview Download
Supplementary_Figure_10_B_BN_PAGE.jpg md5:5054871ed8ae04e9300a55ef93057e4e	81.8 kB	Preview Download
Supplementary_Figure_10_B_DCP.tif md5:9fd322a96e38de14fd45163fc22c889a	175.0 kB	Preview Download
Supplementary_Figure_10_B_HSP60.tif md5:8249dc8a4c6bcbe96056cdd68e671768	569.7 kB	Preview Download
Supplementary_Figure_10_B_OPA.tif md5:f1cc9cbb00ee31b3c9baf9092030eda5	179.2 kB	Preview Download
Supplementary_Figure_10_B_pUb.png md5:8a9d23aa2b6f4bca28bf765252d8c524	152.9 kB	Preview Download
Supplementary_figure_10A_autoradiograph.tif md5:38eecc292a8cb71ef415734249d31fcf	15.4 MB	Preview Download
Supplementary_Figure_10A_Comassie.tif md5:d0e725082213563e3bc5811df67557e7	8.3 MB	Preview Download
Supplementary_Figure_10B_BN_PAGE.jpg md5:5054871ed8ae04e9300a55ef93057e4e	81.8 kB	Preview Download
Supplementary_Figure_10B_DCP.tif md5:9fd322a96e38de14fd45163fc22c889a	175.0 kB	Preview Download
Supplementary_Figure_10B_HSP60.tif md5:eb87907219bd9d7d8edd513b466f2d26	623.2 kB	Preview Download
Supplementary_Figure_10B_OPA.tif md5:f1cc9cbb00ee31b3c9baf9092030eda5	179.2 kB	Preview Download
Supplementary_Figure_10B_pUb.png md5:8a9d23aa2b6f4bca28bf765252d8c524	152.9 kB	Preview Download
Supplementary_Figure_1_PINK1_high.tif md5:6d767928312910dd13b2afcc89ea67aa	750.8 kB	Preview Download
Supplementary_Figure_1_PINK1_low.tif md5:644eaf79e3c4dc4b548679550d3f7ece	265.8 kB	Preview Download
Supplementary_Figure_1_pS65 Ubiquitin_HSP60.tif md5:bd9d65baa22f032cf894c2c3c6fad1f0	384.1 kB	Preview Download
Supplementary_Figure_5A.png md5:2b2ca5c7b28527675db98f10b034b2fa	3.6 MB	Preview Download
Supplementary_Figure_7B.png md5:aadd939a38dab9732343c211e9192f06	1.5 MB	Preview Download
Supplementary_Figure_9A_ Ub.tif md5:72be6c1e693ce141ec881f81c7740dac	207.4 kB	Preview Download
Supplementary_Figure_9A_coomassie.tif md5:88df5f1b49f2cec7c17ca0059d584b67	4.8 MB	Preview Download
Supplementary_Figure_9A_PINK1.tif md5:6a3391abd6181c41d58df936f137530b	1.9 MB	Preview Download
Supplementary_Figure_9A_pS65Ub.tif md5:4bd55245c71ee46bb05e277956841189	2.7 MB	Preview Download
Supplementary_Figure_9B_coomassie.tif md5:56a9dd3868238cc8670d4d2674ca6520	7.4 MB	Preview Download
Supplementary_Figure_9B_PINK1.tif md5:3f42f533fcfcff480956dd864b6dcee6	4.1 MB	Preview Download
Supplementary_Figure_9B_pS65Ub.tif md5:b52241f0603e784ee0fc185e282113c3	1.3 MB	Preview Download
Supplementary_Figure_9B_Ub.tif md5:7df3ede6c23ca35c3f7ec1547a05b9d7	38.4 MB	Preview Download

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Mapping of a N-terminal -helix domain required for human PINK1 stabilisation, Serine228 autophosphorylation and activation in cells

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Figure_1B_GAPDH.tif

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