Published April 10, 2023
| Version v1
Journal article
Open
Regulation of Human PINK1 ubiquitin kinase by Serine167, Serine228 and Cysteine412 phosphorylation
Creators
- Andrew D. Waddell
- Hina Ojha
- Shalini Agarwal
- Christopher J. Clarke
- Ana Terriente-Felix
- Houjiang Zhou
- Poonam Kakade
- Axel Knebel
- Andrew M. Shaw
- Robert Gourlay
- Joby Varghese
- Renata F. Soares
- Rachel Toth
- Thomas Macartney
- Patrick A. Eyers
- Nick Morrice
- Richard Bayliss
- Alexander J. Whitworth
- Claire E. Eyers
- Miratul M. K. Muqit1
Description
Loss-of-function mutations in the human PINK1 kinase (hPINK1) are causative of early-onset Parkinson’s disease (PD). Activation of hPINK1 induces phosphorylated ubiquitin to initiate removal of damaged mitochondria by autophagy. Previously we solved the structure of the insect PINK1 orthologue, Tribolium castaneum PINK1, and showed that autophosphorylation of Ser205 was critical for ubiquitin interaction and phosphorylation (Kumar, Tamjar, Waddell et al., 2017). In this advance, we report new findings on the regulation of hPINK1 by phosphorylation. We reconstitute E. coli expressed hPINK1 activity in vitro by direct incorporation of phosphoserine at the equivalent site Serine 228 (pSer228), providing direct evidence for a role for Ser228 phosphorylation in hPINK1 activation. Furthermore, using mass spectrometry, we identify six novel Ser/Thr autophosphorylation sites including regulatory Serine167 phosphorylation (pSer167), which in addition to pSer228 is required for ubiquitin recognition and phosphorylation. Strikingly, we also detect phosphorylation of a conserved Cysteine412 (pCys412) residue in the hPINK1 activation segment. Structural modelling suggests that pCys412 inhibits ubiquitin recognition and we demonstrate that mutation of Cys412 to Ala renders hPINK1 more active towards ubiquitin when expressed in human cells. These results outline new insights into hPINK1 activation by pSer167 and pSer228 and a novel inhibitory mechanism mediated by pCys412. These findings will aid in the development of small molecule activators of hPINK1.
Description of files:
Figure 1
- Figure 1c OPA1 Coomassie:
- First gel: +Phos Tag
- Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1
- Second gel: -Phos Tag
- Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1
- First gel: +Phos Tag
- Figure 1c blot:
- Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1
- Figure 1d Coomassie:
- Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D
- Figure 1d autorad:
- Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D
- Figure 1f FLAG:
- Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP)
- Figure 1f HSP60:
- Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP)
- Figure 1f OPA1:
- Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP)
- Figure 1f pS228:
- Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP)
- Figure 1f PINK1 pS228 FLAG merge:
- Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP)
- Figure 1g PINK1 pS228 PINK1 multiplex:
- Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A)
- Figure 1g pS228:
- Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A)
- Figure 1g PINK1:
- Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A)
- Figure 1g Tom20:
- Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A)
- Figure 1h HSP60:
- Upper blot
- Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP)
- Lower blot
- Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP), Rest of the lanes: Extra samples not included in the paper.
- Upper blot
- Figure 1h OPA1:
- Upper blot
- Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP)
- Lower blot
- Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP)
- Upper blot
- Figure 1h pS228 PINK1:
- Upper blot
- Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP)
- Lower blot
- Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP)
- Upper blot
- Figure 1h pUb:
- Upper blot
- Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP)
- Lower blot
- Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP)
- Upper blot
- Figure 1- Figure Supplement 2b:
- Recombinant jason chin post-purification. 2 lanes loaded per construct, one for amylose purification and another for the subsequent 6His purification. Lane1 and Lane5 are included in the paper. Lane 1 - WT PINK1, amylose (included in paper), Lane 2 - WT PINK1, 6His, Lane 3 - KI PINK1, amylose, Lane 4 - KI PINK1, 6His, Lane 5 - pSer228 PINK1, amylose (included in paper), Lane 6 - pSer228 PINK1, 6His, Lane 7 - pSer284 PINK1, amylose, Lane 8 - pSer284 PINK1, 6His, Lane 9 - pSer402 PINK1, amylose, Lane 10 - pSer402 PINK1, 6His
- Figure 1- Figure Supplement 2c:
- Lane10 and Lane11 are included in the paper. Lane 1 - 2mg/ml BSA, Lane 2 - 1mg/ml BSA, Lane 3 - 0.5mg/ml BSA, Lane 4 - 0.25mg/ml BSA, Lane 5 - 0.125 mg/ml BSA, Lane 6 – ladder, Lane 7 - unrelated PINK1 construct (Ser228A), Lane 8 - unrelated PINK1 construct (Ser284A), Lane 9 - KI PINK1, Lane 10 - WT PINK1, Lane 11 - pSer228 PINK1, 6His, Lane 12 - pSer284 PINK1, 6His
- Figure 1- Figure Supplement 2d (+ phos tag):
- Lane 1-2: WT recombinant PINK1, Lane 3-4: KI recombinant PINK1, Lane 5-6: pS228 recombinant PINK1, Lane 7-8: pSer284 recombinant PINK1 (Not included in the paper)
- Figure 1- Figure Supplement 2d (no phos tag):
- Lane 1-2: WT recombinant PINK1, Lane 3-4: KI recombinant PINK1, Lane 5-6: pS228 recombinant PINK1, Lane 7-8: pSer284 recombinant PINK1 (Not included in the paper)
- Figure 1- Figure Supplement 2f Coomassie:
- Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D
- Figure 1 - Figure Supplement 2f autorad:
- Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D
- Figure 1 - Figure Supplement 3:
- Immunofluorescence images as mentioned in the paper
Figure 2
- Figure 2a FLAG:
- Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP)
- Figure 2a HSP60:
- Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP), rest of the lanes: Extra samples not included in the paper
- Figure 2a OPA1:
- Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP)
- Figure 2a Phostag:
- Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP)
- Figure 2- Figure Supplement 1a:
- Lane 1: Marker, Lane 3: emp 3-FLAG (-CCCP), Lane 4: emp 3-FLAG (+CCCP), Lane 5: KI PINK1 3-FLAG (-CCCP), Lane 6: KI PINK1 3-FLAG (+CCCP), Lane 7: WT PINK1 3-FLAG (-CCCP), Lane 8: WT PINK1 3-FLAG (+CCCP), Rest of the lanes: Not included in the paper
- Figure 2- Figure Supplement 1b:
- Lane 1: Marker, Lane3-5: 1% TX-100 (Total, soluble, insoluble), Lane 6-8: 1% SDS (Total, soluble, insoluble), Lane 10-12: 2% SDS (Total, soluble, insoluble)
- Figure 2- Figure Supplement 1c:
- Lane 1: Marker, Lane3-5: WT DMSO (Input, SN, IP), Lane 6-8: WT CCCP (Input, SN, IP), Lane 10-12: KI DMSO (Input, SN, IP), Lane 14-16: KI CCCP (Input, SN, IP)
- Figure 2- Figure Supplement 1d DCP:
- Upper blot
- Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP)
- Lower blot
- Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP)
- Upper blot
- Figure 2- Figure Supplement 1d ps228:
- Upper blot
- Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP)
- Lower blot
- Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP)
- Upper blot
- Figure 2- Figure Supplement 1d mix:
- Upper blot
- Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP)
- Lower blot
- Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP)
- Upper blot
- Figure 2- Figure Supplement 1d HSP60 KO:
- Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP)
- Figure 2- Figure Supplement 1d HSP60 WT:
- Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP)
- Figure 2- Figure Supplement 1d ACSL1 HSP60 KO:
- HSP60 blot (Lower band) re-probed for ACSL1 (Upper band); Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP)
- Figure 2- Figure Supplement 1d ACSL1 HSP60 WT:
- HSP60 blot (Lower band) re-probed for ACSL1 (Upper band); Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP)
- Figure 2- Figure Supplement 1e:
- Lane 1: Marker, Lane 3: WT Trypsin (-CCCP), Lane 5: WT chymotrypsin (-CCCP), Lane 7: WT Trypsin (+CCCP), Lane 9: WT chymotrypsin (+CCCP), Lane 11: KI Trypsin (-CCCP), Lane 13: KI chymotrypsin (-CCCP), Lane 15: KI Trypsin (+CCCP), Lane 17: KI chymotrypsin (+CCCP)
Figure 3
- Figure 3c OPA1 PINK1:
- Upper blot: OPA1
- Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A)
- Lower blot: PINK1
- Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A)
- Upper blot: OPA1
- Figure 3c ps228 PINK1:
- Upper blot: pS228 PINK1
- Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A)
- Lower blot: HSP60
- Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A)
- Upper blot: pS228 PINK1
- Figure 3c pUb:
- Blot: pS65 Ub
- Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A)
- Blot: pS65 Ub
Figure 4
- Figure 4e PINK1:
- Full blot (cut according to kDa before probing with antibody); Lane 1: Marker, Lane 3: emp 3FLAG (-CCCP), Lane4: emp 3FLAG (+CCCP for 180 min), Lane 5: KI PINK 3FLAG (-CCCP), Lane6: KI PINK 3FLAG (+CCCP for 180 min), Lane 7: WT PINK 3FLAG (-CCCP), Lane 8: WT PINK 3FLAG (+CCCP for 15 min), Lane 9: WT PINK 3FLAG (+CCCP for 30 min), Lane 10: WT PINK 3FLAG (+CCCP for 45 min), Lane 11: WT PINK 3FLAG (+CCCP for 60 min), Lane 12: WT PINK 3FLAG (+CCCP for 120 min), Lane 13: WT PINK 3FLAG (+CCCP for 180 min), Lane 14: C412A PINK 3FLAG (-CCCP), Lane 15: C412A PINK 3FLAG (+CCCP for 15 min), Lane 16: C412A PINK 3FLAG (+CCCP for 30 min), Lane 17: C412A PINK 3FLAG (+CCCP for 45 min), Lane 18: C412A PINK 3FLAG (+CCCP for 60 min), Lane 19: C412A PINK 3FLAG (+CCCP for 120 min), Lane 20: C412A PINK 3FLAG (+CCCP for 180 min)
- Figure 4e GAPDH OPA:
- Upper bands: OPA1, Lower bands: GAPDH; Lane 1: Marker, Lane 3: emp 3FLAG (-CCCP), Lane4: emp 3FLAG (+CCCP for 180 min), Lane 5: KI PINK 3FLAG (-CCCP), Lane6: KI PINK 3FLAG (+CCCP for 180 min), Lane 7: WT PINK 3FLAG (-CCCP), Lane 8: WT PINK 3FLAG (+CCCP for 15 min), Lane 9: WT PINK 3FLAG (+CCCP for 30 min), Lane 10: WT PINK 3FLAG (+CCCP for 45 min), Lane 11: WT PINK 3FLAG (+CCCP for 60 min), Lane 12: WT PINK 3FLAG (+CCCP for 120 min), Lane 13: WT PINK 3FLAG (+CCCP for 180 min), Lane 14: C412A PINK 3FLAG (-CCCP), Lane 15: C412A PINK 3FLAG (+CCCP for 15 min), Lane 16: C412A PINK 3FLAG (+CCCP for 30 min), Lane 17: C412A PINK 3FLAG (+CCCP for 45 min), Lane 18: C412A PINK 3FLAG (+CCCP for 60 min), Lane 19: C412A PINK 3FLAG (+CCCP for 120 min), Lane 20: C412A PINK 3FLAG (+CCCP for 180 min)
- Figure 4e pUb:
- Lane 1: Marker, Lane 3: emp 3FLAG (-CCCP), Lane4: emp 3FLAG (+CCCP for 180 min), Lane 5: KI PINK 3FLAG (-CCCP), Lane6: KI PINK 3FLAG (+CCCP for 180 min), Lane 7: WT PINK 3FLAG (-CCCP), Lane 8: WT PINK 3FLAG (+CCCP for 15 min), Lane 9: WT PINK 3FLAG (+CCCP for 30 min), Lane 10: WT PINK 3FLAG (+CCCP for 45 min), Lane 11: WT PINK 3FLAG (+CCCP for 60 min), Lane 12: WT PINK 3FLAG (+CCCP for 120 min), Lane 13: WT PINK 3FLAG (+CCCP for 180 min), Lane 14: C412A PINK 3FLAG (-CCCP), Lane 15: C412A PINK 3FLAG (+CCCP for 15 min), Lane 16: C412A PINK 3FLAG (+CCCP for 30 min), Lane 17: C412A PINK 3FLAG (+CCCP for 45 min), Lane 18: C412A PINK 3FLAG (+CCCP for 60 min), Lane 19: C412A PINK 3FLAG (+CCCP for 120 min), Lane 20: C412A PINK 3FLAG (+CCCP for 180 min)
- Figure 4e pS228 PINK1:
- Full blot (cut according to kDa before probing with antibody); Lane 1: Marker, Lane 3: emp 3FLAG (-CCCP), Lane4: emp 3FLAG (+CCCP for 180 min), Lane 5: KI PINK 3FLAG (-CCCP), Lane6: KI PINK 3FLAG (+CCCP for 180 min), Lane 7: WT PINK 3FLAG (-CCCP), Lane 8: WT PINK 3FLAG (+CCCP for 15 min), Lane 9: WT PINK 3FLAG (+CCCP for 30 min), Lane 10: WT PINK 3FLAG (+CCCP for 45 min), Lane 11: WT PINK 3FLAG (+CCCP for 60 min), Lane 12: WT PINK 3FLAG (+CCCP for 120 min), Lane 13: WT PINK 3FLAG (+CCCP for 180 min), Lane 14: C412A PINK 3FLAG (-CCCP), Lane 15: C412A PINK 3FLAG (+CCCP for 15 min), Lane 16: C412A PINK 3FLAG (+CCCP for 30 min), Lane 17: C412A PINK 3FLAG (+CCCP for 45 min), Lane 18: C412A PINK 3FLAG (+CCCP for 60 min), Lane 19: C412A PINK 3FLAG (+CCCP for 120 min), Lane 20: C412A PINK 3FLAG (+CCCP for 180 min)
- Figure 4 - Figure Supplement 1b HSP60:
- Lane 1: WT PINK1 3FLAG (-CCCP), Lane 2: WT PINK1 3FLAG (+CCCP), Lane3: KI PINK1 3FLAG (+CCCP). Rest of the lanes: Not included in the paper
- Figure 4 - Figure Supplement 1b OPA-1:
- Lane 1: WT PINK1 3FLAG (-CCCP), Lane 2: WT PINK1 3FLAG (+CCCP), Lane3: KI PINK1 3FLAG (+CCCP). Rest of the lanes: Not included in the paper
- Figure 4 - Figure Supplement 1b pS228 PINK1:
- Lane 1: WT PINK1 3FLAG (-CCCP), Lane 2: WT PINK1 3FLAG (+CCCP), Lane3: KI PINK1 3FLAG (+CCCP). Rest of the lanes: Not included in the paper
- Figure 4 - Figure Supplement 1b PINK1:
- Lane 1: WT PINK1 3FLAG (-CCCP), Lane 2: WT PINK1 3FLAG (+CCCP), Lane3: KI PINK1 3FLAG (+CCCP). Rest of the lanes: Not included in the paper
- Figure 4 - Figure Supplement 1c commasie WT:
- Lane 1-4: WT PINK1 3FLAG (-CCCP), Lane 6-9: WT PINK1 3FLAG (+CCCP)
- Figure 4 - Figure Supplement 1c commasie KI:
- Lane 1: Marker, Lane 13-16: KI PINK1 3FLAG (+CCCP), Rest of the lanes: Not included in the paper
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md5:73dac2dcefee1c15806e45632df59ce5
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5.6 MB | Preview Download |
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md5:fe2564601acd34f6bcebbff4d854b9e5
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1.3 MB | Preview Download |
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md5:61e7cff0bd817f6b2b7b4a7c0773ce77
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121.5 kB | Preview Download |
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md5:a761636629ba93c00ad6dc7ad202d281
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843.8 kB | Preview Download |
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md5:88945a2b44d12d6b7a7c374fa4e63c62
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229.9 kB | Preview Download |
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md5:94384f3ea9e9c0bb7acf298cecc5e85d
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214.8 MB | Preview Download |
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md5:740cbacd673da9eed1d6b1312e4e57ce
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3.5 MB | Preview Download |
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md5:73dac2dcefee1c15806e45632df59ce5
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5.6 MB | Preview Download |
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md5:a4cb4069e00eebbc6cbb61d5fa68bb7a
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15.7 MB | Preview Download |
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md5:1d770b63ad38db76e750b55fc9e6ad30
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721.6 kB | Preview Download |
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md5:4f820cb03ccad4714c8f8322ac780f2b
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1.5 MB | Preview Download |
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md5:fe2564601acd34f6bcebbff4d854b9e5
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1.3 MB | Preview Download |
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md5:830376d73624d1f52e8bfbae06b06fa5
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14.0 MB | Preview Download |
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md5:e730a10a404c7f9ac083ac69a87cf0c3
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7.7 MB | Preview Download |
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md5:f79137f5ee9bc697ae95bcaf1b81f267
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7.7 MB | Preview Download |
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md5:be6e86b5105df26fe4d7b838954a52c1
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7.7 MB | Preview Download |
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md5:922217223c4b17121eb1f06798ac63ad
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3.0 MB | Preview Download |
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md5:423c9a19157f07a76ce47a21a57a5bd3
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666.0 kB | Preview Download |
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md5:650601994f95040f422265d63090c38c
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1.3 MB | Preview Download |
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md5:137ae7e2056ce440a1c9d8c545936967
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37.6 MB | Preview Download |
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md5:bf128e60ff6d3efef156c4ab37da6bbb
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1.7 MB | Preview Download |
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md5:ada5782004119953c80b81d8bd07b4f1
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2.0 MB | Preview Download |
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md5:d737417b382da11b7fa6f391273ab96e
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2.8 MB | Preview Download |
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md5:3efd6b0b92f60a42a81c6d3963c79a84
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3.3 MB | Preview Download |
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md5:b3340557ea094db30b9209d5325ab6b7
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487.7 kB | Preview Download |
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md5:d572d97f7da34350c96a92be1e6a4c30
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9.4 MB | Preview Download |
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md5:238c46b1e29cc47b42ec3b0fefb49b54
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237.1 kB | Preview Download |
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md5:45095f6cf2a12c448f3de034813fc91f
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424.4 kB | Preview Download |
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md5:77308809050266c4bbf1daf65cc49987
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548.7 kB | Preview Download |
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md5:35afc249fc0d2badb7cd6874dbce38a7
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188.7 kB | Preview Download |
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md5:800d9f31416ebe1d58a0a63345cbb538
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312.1 kB | Preview Download |
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md5:f067592bf8bc8e5721508ab055c4ca71
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3.4 MB | Preview Download |
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md5:ab1d05e2efae81c03b5fa4a00b94d692
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1.6 MB | Preview Download |
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md5:e2a3403e345289fa3c20846e265de1ec
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1.4 MB | Preview Download |
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md5:f067592bf8bc8e5721508ab055c4ca71
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3.4 MB | Preview Download |
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md5:c1bb0ee2d1c6eb48fdd3dac61b051cda
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907.6 kB | Preview Download |
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md5:75ee459dba130f0e030ff51fc9c849f4
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350.0 kB | Preview Download |
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md5:e2697b9cf84d6cc8ed51481ecef81bac
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14.1 MB | Preview Download |
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md5:5dd83653aaa3fe1d923dd5588065f02d
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609.1 kB | Preview Download |
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md5:21b30df14bfb0e57e971fb069b0ad2e3
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1.2 MB | Preview Download |
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md5:64ed9a492b0fce1d467450ed3c5bee75
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186.3 kB | Preview Download |
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md5:bdd0f53ab3f5e725aaa9305e468d8c21
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124.6 kB | Preview Download |
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md5:bdd0f53ab3f5e725aaa9305e468d8c21
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124.6 kB | Preview Download |
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md5:4ab96ac8548a845169e1eb8c13d6f1ca
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259.5 kB | Preview Download |
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md5:63dde75495aad90ece336cc85dfc6023
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231.2 kB | Preview Download |
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md5:f34463a2e8b14b17646a948b1b695190
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691.7 kB | Preview Download |
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md5:5a8769b4ef38fc8a3228d2bb75215633
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102.2 kB | Preview Download |
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md5:50f4c5f308608ba213f7a0dcf7923224
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268.4 kB | Preview Download |
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md5:0eeaae51effb92608018c7a525964776
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254.6 kB | Preview Download |
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md5:f943f6f67b06db229eea82108f167cba
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143.2 kB | Preview Download |
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md5:793bc90edc73f7ebf970e259528c3713
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215.3 kB | Preview Download |
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md5:97d5271be6b15c7b8cc6a435d905f5e1
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246.9 kB | Preview Download |
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md5:f6ea875fd340cedc500797edc01fb403
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655.5 kB | Preview Download |
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md5:e46c4155af67f908f9094092bda090ef
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524.1 kB | Preview Download |
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md5:e0fa0d5f95311758403c792245e2998f
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7.6 MB | Preview Download |
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md5:b6e70bc38d999eff4df8b03f0323760b
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7.6 MB | Preview Download |
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md5:d809c6ec18be1a75e1e9f68ebd45e0da
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7.6 MB | Preview Download |
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md5:e3f24c1a2ec7213ec7ae0b61af7f7160
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7.6 MB | Preview Download |
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md5:d9bcce826ad7202bd52e98924c2508a1
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7.6 MB | Preview Download |
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md5:ab8315e6a6515fd6f82d721e4d065bf9
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7.6 MB | Preview Download |
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md5:589902cde8d45bcf9e16400418fc8f13
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7.6 MB | Preview Download |
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md5:fdfbc67947d6ae148d63c8fb72735a04
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7.6 MB | Preview Download |
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md5:46b0ab10e75a9577106d338166d9bf35
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7.6 MB | Preview Download |
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md5:fedf2e7daae76a07c5600a67a99b09db
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7.6 MB | Preview Download |
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md5:659ee2f899b25661ef34c63397d63771
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7.6 MB | Preview Download |
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md5:ab26e99990731740dd442def88773a61
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7.6 MB | Preview Download |
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md5:1b622981ba2c68217f11cc9ed33bfcc0
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7.6 MB | Preview Download |
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md5:86217d4280627558db57944e625b9478
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7.6 MB | Preview Download |
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md5:8a22f2fa20d9c35abb283d6e008a57c5
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7.6 MB | Preview Download |
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md5:ec9b189e4eebe1128d39d7fb07dc4ec2
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7.6 MB | Preview Download |
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md5:04ba410e58ee20d148b563be6e9a6d60
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7.6 MB | Preview Download |
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md5:bc3bedf5dc129e4dc3476298a54be0d0
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7.6 MB | Preview Download |
