Global ubiquitylation analysis of mitochondria in primary neurons identifies endogenous Parkin targets following activation of PINK1
- 1. MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom, DD1 5EH, U.K.
- 2. Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
- 3. MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom, DD1 5EH, U.K.; Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
- 4. Data Analysis Group, Division of Computational Biology, School of Life Sciences, University of Dundee, Dundee, United Kingdom, DD1 5EH, U.K.
- 5. Dundee Imaging Facility, School of Life Sciences, University of Dundee, Dundee, United Kingdom, DD1 5EH, U.K
- 6. Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA; Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Description
How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell lines with few studies in neurons. Herein we have undertaken proteomic analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1 activation-dependent diGLY sites in 22 proteins conserved across mouse and human systems. We employ reconstitution assays to demonstrate direct ubiquitylation by Parkin in vitro. We also identified a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarisation in neurons. Finally we have developed an online resource to search for ubiquitin sites and enzymes in mitochondria of neurons, MitoNUb. These findings will aid future studies to understand Parkin activation in neuronal subtypes.
FILE DECRIPTIONS
Figure 1C: Immunoblots for PINK1 signaling in PINK1 WT and KO mouse cortical neurons.
Scans of X-ray film:
- GAPDH shown in Figure1C_GAPDH.tif
- Parkin shown in Figure1C_Parkin.tif
- Phospho-Ser65 Parkin shown in Figure1C_ParkinPSer65.tif
- PINK1 shown in Figure1C_PINK1.tif (immunoprecipitation)
- Rab8A shown in Figure1C_Rab8A.tif
- Phospho-Ser111 Rab8A shown in Figure1C_Rab8APSer111.tif
- Ubiquitin shown in Figure1C_ S10A_Ubiquitin.tif (same blot used for Figure_S10A, Halo-multiDSK pull-down)
- Phospho-Ser65 Ubiquitin shown in Figure1C_S10A_UbiquitinPSer65.tif (same blot used for Figure_S10A, Halo-multiDSK pull-down)
Figure 4A: Immunoblots for time-course of Parkin-dependent substrates in C57BL/6J neurons.
Scans of X-ray film:
- CISD1 shown in Figure4A_CISD1.tif (Halo-multiDSK pull-down)
- Figure4A_INPUT_CISD1.tif (INPUT)
- CPT1α shown in Figure4A_ CPT1a.tif (Halo-multiDSK pull-down)
- Figure4A_INPUT_CPT1a.tif (INPUT)
- Ubiquitin shown in Figure4A_Ubiquitin.tif (Halo-multiDSK pull-down)
- Figure4A_INPUT_Ubiquitin.tif (INPUT)
- Phospho-Ser65 Ubiquitin shown in Figure4_UbiquitinPSer65.tif (Halo-multiDSK pull-down)
- GAPDH Shown in Figure4A_INPUT_GAPDH.tif (INPUT)
Figure 4B: Immunoblots for validation of Parkin-dependent substrates in PARKIN WT and KO neurons.
Scans of X-ray film:
- CISD1 shown in Figure4B_CISD1.tif (Halo-multiDSK pull-down)
- (bottom blot) Figure4B_INPUT_CPT1a_CISD1.tif (INPUT)
- CPT1α shown in Figure4B_ CPT1a.tif (Halo-multiDSK pull-down)
- (top blot) Figure4B_INPUT_CPT1a_CISD1.tif ( (INPUT)
- Ubiquitin shown in Figure4B_Ubiquitin.tif (Halo-multiDSK pull-down)
- Figure4B_INPUT_Ubiquitin.tif (INPUT)
- Phospho-Ser65 Ubiquitin shown in Figure4B_UbiquitinPSer65.tif (Halo-multiDSK pull-down)
- GAPDH Shown in Figure4B_INPUT_GAPDH.tif (INPUT)
Figure 5A: Immunoblots for in vitro reconstitution assay in PINK1 WT and KO mouse embryonic fibroblasts.
Scans of X-ray film:
- CISD1shown in Figure5A_CISD1.tif
- CPT1α shown in Figure5A_ CPT1a.tif
- CYB5B shown in Figure5A_CYB5B.tif
- HK1 shown in Figure5A_HK1.tif
- MFN2 shown in Figure5A_MFN2.tif
- VDAC shown in Figure5A_VDAC.tif
- Phospho-Ser65 Parkin shown in Figure5A_ParkinPSer65.tif
- Parkin shown in Figure5A_Parkin.tif
- Ubiquitin shown in Figure5A_Ubiquitin.tif
- Phospho-Ser65 Ubiquitin shown in Figure5A_UbiquitinPSer65.tif
Figure 5B: Immunoblots for validation of Parkin-dependent substrates in in vitro studies.
Scans of Western blots (LI-COR):
- CPT1α shown in Figure5B_ CPT1 α_800nm.tif
- Flag-Ub (CPT1α) shown in Figure5B_ CPT1 α_Flag_800nm.tif
- His (CPT1α) shown in Figure5B_ CPT1 α_His_800nm.tif
- Miro1 shown in Figure5B_Miro1_800nm.tif
- Flag-Ub (Miro1) shown in Figure5B_ Miro1_Flag_800nm.tif
- His (Miro1) shown in Figure5B_ Miro1_His_800nm.tif
Figure 5C: Immunoblots for time-course of CPT1α ubiquitylation in in vitro studies.
Scans of Western blots (LI-COR):
- CPT1α shown in top blot: Figure5C_Flag_CPT1α_800nm.tif
- Flag-Ub shown in bottom blot: Figure5C_Flag_CPT1α_800nm.tif
- His shown in Figure5C_ CPT1 α_His_800nm.tif
Figure S3A: Immunoblots for PINK1 signaling in C57BL/6J neurons.
Scans of X-ray film:
- GAPDH shown in FigureS3A_GAPDH.tif
- Parkin shown in FigureS3A_Parkin.tif
- Phospho-Ser65 Parkin shown in FigureS3A_ParkinPSer65.tif
- PINK1 shown in FigureS3A_PINK1.tif (immunoprecipitation)
- Rab8A shown in FigureS3A_Rab8A.tif
- Phospho-Ser111 Rab8A shown in FigureS3A_Rab8APSer111.tif
- Ubiquitin shown in FigureS3A_Ubiquitin.tif (same blot used for Figure_4A, Halo-multiDSK pull-down)
- Phospho-Ser65 Ubiquitin shown in FigureS3A_UbiquitinPSer65.tif (same blot used for Figure_4A, Halo-multiDSK pull-down)
- Phospho-Ser111 Rab8A and Phospho-Ser65 Parkin also shown in FigureS3A_ParkinPSer65_Rab8APSer111.tif
Figure S3B: Immunoblots for comparison of Halo-multiDSK and Halo-TUBE in C57BL/6J neurons.
- Ubiquitin shown in FigureS9_S3_Ubiquitin.tif (same blot used for Figure_S9)
- Phospho-Ser65 Ubiquitin shown in FigureS9_S3_UbiquitinPSer65.tif (same blot used for Figure_S9)
Figure S3C: Immunoblots for PINK1 signaling in Parkin WT and KO mouse cortical neurons.
Scans of X-ray film:
- GAPDH shown in FigureS3C_GAPDH.tif
- Parkin shown in FigureS3C_Parkin.tif
- Phospho-Ser65 Parkin shown in FigureS3C_ParkinPSer65.tif
- PINK1 shown in FigureS3C_PINK1.tif (immunoprecipitation)
- Rab8A shown in FigureS3C_Rab8A.tif
- Phospho-Ser111 Rab8A shown in FigureS3C_Rab8APSer111.tif
- Ubiquitin shown in FigureS3C_ Ubiquitin.tif (same blot used for Figure_4B, Halo-multiDSK pull-down)
- Phospho-Ser65 Ubiquitin shown in FigureS3C_ UbiquitinPSer65.tif (same blot used for Figure_4B, Halo-multiDSK pull-down)
Figure S4: Immunoblots for PINK-Parkin signaling in VPS35 D620N mouse cortical neurons.
Scans of X-ray film:
- GAPDH shown in FigureS4_GAPDH.tif
- Parkin shown in FigureS4_Parkin.tif
- Phospho-Ser65 Parkin shown in FigureS4_ParkinPSer65.tif
- Rab8A shown in FigureS4_Rab8A.tif
- Phospho-Ser111 Rab8A shown in FigureS4_Rab8APSer111.tif
- CISD1 shown in FigureS4_ CISD1.tif (Halo-UBQLN1 pull-down)
- Phospho-Ser65 Ubiquitin shown in FigureS4_ UbiquitinPSer65.tif (Halo-UBQLN1 pull-down)
- VPS35 shown in FigureS4_VPS35.tif
Scan of Memcode shown in FigureS4_Memcode.tif
Figure S6: Immunoblots for biochemical analysis in C56BL/6J mouse cortical neurons.
Scans of X-ray film:
- GAPDH shown in FigureS6_GAPDH.tif
- Phospho-Ser65 Ubiquitin shown in FigureS6_ UbiquitinPSer65.tif
Figure S8: Immunoblots for biochemical analysis in PINK1 WT and KO mouse cortical neurons.
Scans of X-ray film:
- GAPDH shown in FigureS8_GAPDH.tif
- Phospho-Ser65 Ubiquitin shown in FigureS8_ UbiquitinPSer65.tif
Figure S9: Immunoblots for biochemical analysis of ubiquitylated target in C56BL/6J mouse cortical neurons.
Membrane-enriched lysate subjected to ubiquitin capture using UBQLN1(TUBE), multiDSK and mutant multiDSK pull-down (Illustration of sample loading in figures FigureS9_ACSL6,_MFN2, _UbiquitinPSer65, _NAV1.7).
Scans of X-ray film:
- Ubiquitin shown in FigureS9_S3_Ubiquitin.tif (same blot used for Figure_S3B)
- Phospho-Ser65 Ubiquitin shown in FigureS9_S3_UbiquitinPSer65.tif (same blot used for Figure_S3B)
- ABCD3 shown in FigureS9_ABCD3.tif
- ACSL1shown in FigureS9_ ACSL1.tif
- ACSL6 shown in FigureS9_ACSL6.tif
- AGPAT5 shown in FigureS9_AGPAT5.tif
- ARHGAP33 shown in FigureS9_ARHGAP33.tif
- ATAD1 shown in FigureS9_ATAD1.tif
- CAD shown in FigureS9_ CAD.tif
- CAMK2A shown in FigureS9_CAMK2A.tif
- CAMK2B shown in FigureS9_CAMK2B.tif
- CDK16 shown in FigureS9_CDK16.tif
- CISD1shown in FigureS9_CISD1.tif
- CNN3 shown in FigureS9_CNN3.tif
- CPT1α shown in FigureS9_ CPT1A.tif
- CYB5B shown in FigureS9_CYB5B.tif
- CYB5R3 shown in FigureS9_CYB5R3.tif
- DCAKD shown in FigureS9_DCAKD.tif
- DCAMKL2 shown in FigureS9_DCAMKL2.tif
- FAM213A shown in FigureS9_FAM213A.tif
- FBXO41 shown in FigureS9_FBXO41.tif
- GK shown in FigureS9_GK.tif
- HK1 shown in FigureS9_HK1.tif
- HSDL1 shown in FigureS9_HSDL1.tif
- MAO-A shown in FigureS9_MAOA.tif
- MAO-B shown in FigureS9_MAOB.tif
- MAPRE2 shown in FigureS9_MAPRE2.tif
- MARC2 shown in FigureS9_MARC2.tif
- MFN1 shown in FigureS9_MFN1.tif
- MFN2 shown in FigureS9_MFN2.tif
- NAV1.7 shown in FigureS9_NAV1.7.tif
- P23 shown in FigureS9_p23.tif
- PRKCG shown in FigureS9_PRKCG.tif
- RAB5C shown in FigureS9_Rab5c.tif
- RHOT2 shown in FigureS9_RHOT2.tif
- RIMS4 shown in FigureS9_RIMS4.tif
- RUFY3 shown in FigureS9_RUFY3.tif
- SH3BP4 shown in FigureS9_SH3BP4.tif
- SNX3 shown in FigureS9_SNX3.tif
- TDRKH shown in FigureS9_TDRKH.tif
- TOMM70 shown in FigureS9_TOMM70.tif
Figure S10A: Immunoblots for validation of Parkin-dependent substrates in PINK1 WT and KO neurons.
Scans of X-ray film:
- CISD1 shown in Figure S10A _CISD1.tif (Halo-multiDSK pull-down)
- Figure S10A _INPUT_CISD1.tif (INPUT)
- CPT1α shown in Figure S10A _ CPT1A.tif (Halo-multiDSK pull-down)
- Figure S10A _INPUT_ CPT1A.tif (INPUT)
- Ubiquitin shown in Figure1C_ S10A _Ubiquitin.tif (same blot used for FigureS1C, Halo-multiDSK pull-down)
- Figure S10A _INPUT_Ubiquitin.tif (INPUT)
- Phospho-Ser65 Ubiquitin shown in Figure1C_ S10A_UbiquitinPSer65.tif (same blot used for FigureS1C, Halo-multiDSK pull-down)
- GAPDH Shown in Figure S10A _INPUT_GAPDH.tif (INPUT)
Figure S10B: Immunoblots for time-course of Parkin-dependent substrates in SH-SY5Y cells.
Scans of X-ray film:
- CISD1 shown in Figure S10B _CISD1.tif (Halo-multiDSK pull-down)
- CPT1α shown in Figure S10B _ CPT1A.tif (Halo-multiDSK pull-down)
- Ubiquitin shown in Figure S10B _Ubiquitin.tif (Halo-multiDSK pull-down)
- Phospho-Ser65 Ubiquitin shown in Figure S10B _UbiquitinPSer65.tif (Halo-multiDSK pull-down)
- GAPDH Shown in Figure S10B _GAPDH.tif
- Parkin shown in FigureS10B_Parkin.tif
- Phospho-Ser65 Parkin shown in FigureS10B_ParkinPSer65.tif
- PINK1 shown in FigureS10B_PINK1.tif
- OPA1 shown in FigureS10B_OPA1.tif
Figure S11: Immunoblots for in vitro reconstitution assay in HeLa cells.
Scans of X-ray film:
- CISD1shown in FigureS11_CISD1.tif
- CPT1α shown in FigureS11_ CPT1a.tif
- CYB5B shown in FigureS11_CYB5B.tif
- HK1 shown in FigureS11_HK1.tif
- MFN2 shown in FigureS11_MFN2.tif
- VDAC shown in FigureS11_VDAC.tif
- Phospho-Ser65 Parkin shown in FigureS11_ParkinPSer65.tif
- Parkin shown in FigureS11_Parkin.tif
- Ubiquitin shown in FigureS11_Ubiquitin .tif
- Phospho-Ser65 Ubiquitin shown in FigureS11_UbiquitinPSer65 .tif
FigureS12A: Purification of CPT1α protein.
- FigureS12A_ Akta_Purifier_Curves.txt.
Tab delimited text of data from the AKTA system, plotted in figure S12A. Columns are laid out as volume/measured parameter for each parameter from the Akta. The columns plotted in figure S12A were mAU (columns A and B in excel) and fractions (columns M and N in excel).
- Figure_S12A_Coomassie_Gel.tif.
Coomassie stained gels of fractions from the AKTA system in figure S12A.
Top: Left hand Coomassie gel, figure S12A
Bottom: Right hand Coomassie gel, figure S12A
FigureS12B-C: Purification of recombinant Parkin targets
Blots shown in the paper are highlighted in bold.
- FigureS12B_Ub_Targets_High_800nm.tif
Coomassie stained gels of the parkin targets alongside a BSA curve.
Left: Lane: 1: Mwt Marker, 2: Fam213A, 3: MAO-B, 4: CAMK2α, 5: MAO-A (figure S12B), 6: GST-MAPRE2, 7: MBP-CYB5R3, 8: MBP-CYB5B, 9: MBP-CYB5B, 10: SNX3, 11: His-SUMO-MAO-B, 12: GST-MAO-B, 13: 0.03125 ug BSA, 14: 0.0625 ug BSA, 15: 0.125 ug BSA, 16: 0.25 ug BSA, 17: 0.5 ug BSA, 18: 1 ug BSA
Right: Lane: 1: Mwt Marker, 2: GST-MAO-A, 3: GST-FAM213A, 4: MBP-CAMK2α (figure S12B), 5: GST-TDRKH, 6: MBP-CPT1α, 7: MBP-CYB5B, 8: CAMK2β, 9:, 10: His-SUMO-MAO-B, 11: GST-MAO-B, 12: 0.03125 ug BSA, 13: 0.0625 ug BSA, 14: 0.125 ug BSA, 15: 0.25 ug BSA, 16: 0.5 ug BSA, 17: 1 ug BSA
- Figure_S12B_Ub_Targets_Low_800nm.tif
Coomassie stained gels of the parkin targets alongside a BSA curve.
Lane: 1: Mwt Marker, 2: GST-Miro1 (figure S12B), 3: His-SUMO-Fam213A, 4: His-SUMO-MAPRE2, 5: His-SUMO-MAO-A, 6: His-SUMO-MAO-B, 7: His-SUMO-SNX26, 8: His-SNX3, 9: His-MAPRE2, 10: GST-FAM213A, 11: His-SUMO-CAMK2α, 12: His-SUMO-CAMK2β, 13: GST-FAM213A, 14: 0.03125 ug BSA, 15: 0.0625 ug BSA, 16: 0.125 ug BSA, 17: 0.25 ug BSA, 18: 0.5 ug BSA, 19: 1 ug BSA
- Figure_S12C_Ub_Targets_800nm.tif
Coomassie stained gels of the parkin targets in duplicate alongside a BSA curve.
Top: Lane: 1: Mwt Marker, 2+3: ACSL1 45-end (Figure S12C), 4+5: SNX3 (Figure S12C), 6+7: MFN1, 8+9: MFN2, 10+11: 0.125 ug BSA, 12+13: 0.25 ug BSA, 14+15: 0.5 ug BSA, 16+17: 1 ug BSA
Middle: Lane: 1: Mwt Marker, 2+3: CAMK2α, 4+5: CAMK2β (Figure S12C), 6+7: MAO-A, 8+9: MAO-B (Figure S12C), 10+11: 0.125 ug BSA, 12+13: 0.25 ug BSA, 14+15: 0.5 ug BSA, 16+17: 1 ug BSA
Bottom: Lane: 1: Mwt Marker, 2+3: SRCIN, 4+5: His-MAPRE2, 6+7: FAM213A (Figure S12C), 8+9: CISD1, 10+11: 0.125 ug BSA, 12+13: 0.25 ug BSA, 14+15: 0.5 ug BSA, 16+17: 1 ug BSA
FigureS13-S14: Immunoblots for validation of Parkin-dependent substrates in in vitro studies.
Blots shown in the paper are highlighted in bold.
Scans of X-ray film:
- FigureS13A-B_S14C-D_a6His_Film.tif
Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1
Membranes from left to right:
Top: 1: FAM213A (Figure S13B), 2: MAO-B (Figure S13A), 3: MAPRE2, 4: CAMK2β (Figure S14D)
Top: 1: CAMK2α (Figure S14C), 2: Miro1, 3: MAO-A (Figure S13A)
- FigureS13A-B_S14C-D_aFLAG_Film.tif
Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1
Membranes from left to right:
Top: 1: FAM213A (Figure S13B), 2: MAPRE2, 3: MAO-A (Figure S13A)
Top: 1: MAO-B (Figure S13A), 2: CAMK2α (Figure S14C), 3: CAMK2β (Figure S14D), 4: Miro1
- FigureS13A-B_S14C-D_aTarget_05m00s_Film.tif
Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1
Membranes from left to right:
Top: 1: FAM213A, 2: MAPRE2, 3: MAO-A
Top: 1: MAO-B, 2: CAMK2α (Figure S14C), 3: CAMK2β (Figure S14D), 4: Miro1
- FigureS13A-B_S14C-D_aTarget_06m00s_Film.tif
Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1
Membranes from left to right:
Top: 1: FAM213A (Figure S13B), 2: MAPRE2, 3: MAO-A (Figure S13A)
Top: 1: MAO-B (Figure S13A), 2: CAMK2α, 3: CAMK2β, 4: Miro1
FigureS13C: Immunoblots for validation of Parkin-dependent substrates in in vitro studies.
Scans of Western blots (LI-COR):
- ACSL1 shown in FigureS13C_ACSL1_800nm.tif
- Flag-Ub (ACSL1) shown in Figure_S13C_ACSL1_Flag_800nm.tif
- His (ACSL1) shown in Figure_S13C_ACSL1_His_800nm.tif
FigureS14B: Immunoblots for validation of Parkin-dependent substrates in in vitro studies.
Blots shown in the paper are highlighted in bold.
Scans of Western blots (LI-COR):
- SNX3 shown in FigureS14B_SNX3_800nm.tif
Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1
Membranes, left to right: SNX3 (Figure S14B), FAM213A, Miro1, Miro1
- Flag-Ub (SNX3) shown in Figure_S14B_Flag_SNX3_800nm.tif
Gel loading: lanes: 1+2: – PINK1, 3+4:WT PINK1, 5+6: KD PINK1
Membranes, left to right: Miro1, FAM213A, Miro1, SNX3 (Figure S14B)
- His (SNX3) shown in Figure_S14B_His_SNX3_700nm.tif
Gel loading: lanes: 1+2: – PINK1, 3+4: WT PINK1, 5+6: KD PINK1
Membranes, left to right: CPT1α, MFN1, SNX3 (Figure S14B), FAM213A
FigureS1: Immunocytochemistry of mouse cortical neurons.
- FigureS1_TILE_maximun intensity projection.tif
Original acquisition: MAP2 neuronal marker (green-488); GFAP astrocytic marker (red-594); Hoechst nuclear marker (blue 361/497).
- FigureS1MGB (RGB).tif
Modified from FigureS1_TILE_maximun intensity projection.tif: MAP2 (green); GFAP (magenta); Hoechst (blue).
Table for Fig1DE.xlsx
Numerical data for the charts shown in Figure1D and Figure1E.
Table for FigS1A.xlsx
Numerical data for quantification of Map2 and GFAP in mouse cortical neurons shown in FigureS1A.
Table for FigS1B (Cortical neurons_DIA_Report).xlsx
Numerical data for DIA proteomic analysis of AO and DMSO treated cortical neurons shown in FigureS1B.
Table for FigS2B_C_Oxygraph.xlsx
Numerical data for the charts shown in FigureS2B and FigureS2C.
Table for Fig3A.xlsx
Numerical data for total protein abundance in PINK1 WT and KO shown in Figure3A.
Table for Fig3B.xlsx
Numerical data of abundance of phosphorylated-Ser65 or Ser57 shown in Figure3B.
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Figure1C_GAPDH.tif
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md5:b2dce520f6d459b33dff176c14dca471
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5.6 MB | Preview Download |
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md5:45a9b8461c3ce811473bde01ec12484d
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164.9 MB | Preview Download |
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md5:808340ebefe2f02982916c8a7181b5fa
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115.5 MB | Preview Download |
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md5:aae209b961cb943f3d9f67144fe21269
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125.5 MB | Preview Download |
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md5:1b7a108838448d6503f0bcc8c66cd361
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241.0 MB | Preview Download |
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md5:0d56ef973909c0b5bef8cc7526455abb
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76.8 MB | Preview Download |
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md5:4dd34230bebd8abdc45e8d4e84eb8c77
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261.1 MB | Preview Download |
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md5:c6b247dad1dd4db176f5e0e1b91f9c03
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222.3 MB | Preview Download |
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md5:810688079e81da6339940875804bd210
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99.3 MB | Preview Download |
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md5:dda6a2e8b08a133e7d8196dcb34f154c
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14.8 MB | Preview Download |
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md5:474e2e86f2bef8c13ad3bc2aea22bd06
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16.0 MB | Preview Download |
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md5:c1315951b2de8eed102270f76d4615c8
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13.1 MB | Preview Download |
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md5:9ef7888a45deeae3f93af8d111a8dfd5
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12.5 MB | Preview Download |
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md5:879735921e95d3d1389c0b7d5e1fcf3d
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11.2 MB | Preview Download |
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md5:677bd8e8c46b2c154a7d74546c8af15b
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13.9 MB | Preview Download |
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md5:b5dc04b9f3469c02081459d026775abb
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23.5 MB | Preview Download |
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md5:600a8952f035a348c473c3df92d1eaf2
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24.1 MB | Preview Download |
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md5:903cca83b9d73c1ddb2e82bb0c721709
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15.1 MB | Preview Download |
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md5:02eb7a1b364edfd9537edc7794c822f4
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16.6 MB | Preview Download |
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md5:6b82b7e3be2aa144b243e8b6990ff7a6
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17.0 MB | Preview Download |
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md5:c237762f0013e4b95cc02a00d8de8eb3
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20.2 MB | Preview Download |
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md5:eb5e0e8b27c097837b38b07f075f54f0
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16.3 MB | Preview Download |
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md5:31d090c36e8335543d00363b13b3a5db
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23.7 MB | Preview Download |
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md5:7f4d20f45818cdc0e65ecc63c01c03ad
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16.0 MB | Preview Download |
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md5:c8b134042cce094331e82239db283bd0
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3.4 MB | Preview Download |
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md5:fb4afede8cbd988e8ff82b76b436eb57
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3.5 MB | Preview Download |
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md5:7610f9769428028bc02e9b75ef85f7b5
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3.6 MB | Preview Download |
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md5:acbdfbe510992ae95eef89926a8d33d0
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3.0 MB | Preview Download |
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md5:bc5dc513f0d5cc5aa67830f7c923077b
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3.0 MB | Preview Download |
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md5:7cab795ff6c7c156033b78f182ffe312
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3.3 MB | Preview Download |
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md5:72b771d5e5bdbe174bdc2fc3b14aed06
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3.3 MB | Preview Download |
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md5:d101843933f4bf266725aad336efa46d
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5.8 MB | Preview Download |
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md5:873704368ca405be52b1ef18a44f8237
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6.0 MB | Preview Download |
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md5:0f5367a0300619cc75b4b63f9458d3a4
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2.8 MB | Preview Download |
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md5:8d2b745abdfd3fd32cbcfd4c3c1ed721
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189.3 kB | Download |
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md5:2485f4000aeb045569fddfb76dd4c22a
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11.2 MB | Preview Download |
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md5:c6a329c4010515cfe0d4440a9c4ce38d
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188.0 MB | Preview Download |
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md5:1eec227a0034b37ad644080bc5d2333c
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78.3 MB | Preview Download |
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md5:687154c9a78d0b3b04e208bf3317f4c5
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208.8 MB | Preview Download |
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md5:669d9e47d30e3ed9c88dcc9a6fc4a80b
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32.2 MB | Preview Download |
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md5:927831b3cac9dde8920fad7a70a57642
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31.3 MB | Preview Download |
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md5:db27be34075fd3e4bec1db24c7fd4745
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30.8 MB | Preview Download |
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md5:48a3e0e6d3ad46d95f36ac66ab60600c
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30.8 MB | Preview Download |
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md5:531ca6969f7febfee5e637477d99514a
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101.4 MB | Preview Download |
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md5:85ec9499560fb7a41a17ef056aaef14d
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129.7 MB | Preview Download |
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md5:1d728d67f1eab299aee53fb3eb348a27
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102.5 MB | Preview Download |
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md5:a1bcd2cb56a60710de5aaa606e0573f7
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256.9 MB | Preview Download |
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md5:82a6c4172d171880ce78854ebeb569f8
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26.8 MB | Preview Download |
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md5:b1e111a9821ba7c5348f727834e1e641
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123.3 MB | Preview Download |
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md5:9a23903d1d1e6a60d96fe4e2cdb26129
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75.0 MB | Preview Download |
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md5:382e617f28cb7c79ccb723dbf1e0d222
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75.0 MB | Preview Download |
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md5:756cf4a21f9a331846ddd457a3e192e3
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2.9 MB | Preview Download |
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md5:35aecf8ededaf896e82c8fec85d1a94b
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3.0 MB | Preview Download |
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md5:a98f68481ce69b8572cf487e56035439
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2.3 MB | Preview Download |
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md5:30cdfbee2cf1853cd6ea45ee99f83d5f
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10.8 MB | Preview Download |
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md5:0124994fef03bc7abb3d28ea53114bf7
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12.2 MB | Preview Download |
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md5:19f4c82a81b2b9f0297f2a54b7fec9f4
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3.6 MB | Preview Download |
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md5:ffc43b451404e31c601cb0f5eb4f1366
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2.1 MB | Preview Download |
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md5:9862f07af63739a41ee9bb734987b14a
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10.2 MB | Preview Download |
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md5:36ddca6eda029edf5ee09a80dcf56aad
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12.4 MB | Preview Download |
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md5:b0a15e65c8eaf945cfdc04932f1e4dd2
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4.0 MB | Preview Download |
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md5:aeec4662abe7cc27855737e5978a2168
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4.8 MB | Preview Download |
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md5:4f4f4c0cc14a4d7bf48cb6f1ce8816cd
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2.7 MB | Preview Download |
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md5:634399e9b11f4797a838863867913b2b
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9.1 MB | Preview Download |
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md5:79754fed0e62c288b667828a61b82b22
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10.2 MB | Preview Download |
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md5:c806a5b72ae01186336eeee37a584d18
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7.2 MB | Preview Download |
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md5:6b3acaa8a8cbd8af1a3a9e418b066375
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11.7 MB | Download |
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md5:d7795b280c77620cf60f360027297941
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11.8 MB | Preview Download |
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md5:e860e8c9d0cc9ce5e8803fef2fef675e
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4.6 MB | Preview Download |
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md5:d075ed6e64c2799a4fe91ad34d1e7ea5
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6.4 MB | Preview Download |
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md5:1c54d761f6cf952bd807c8ea03bbec53
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13.4 MB | Preview Download |
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md5:f8c10df8d7af82bdfee436deb2c42605
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4.9 MB | Preview Download |
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md5:0e12c1be6bf09ac33603fe90ae909a2b
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4.8 MB | Preview Download |
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md5:f5870a58fb3b5c60921e6c1823286d42
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10.8 MB | Preview Download |
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md5:b49876ca44791fdcbc322f2146a7d657
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12.5 MB | Preview Download |
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md5:c2edb6562e8bb9894b2768054aefbbed
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5.0 MB | Preview Download |
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md5:e4828a06a91de96b7cebcb92fd717267
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5.4 MB | Preview Download |
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md5:0066e8fa15bfee86a2b8c518d3cf4f63
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8.4 MB | Preview Download |
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md5:43ca3bcf7e854a7252f857d93f0c3034
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7.8 MB | Preview Download |
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md5:fc99e8d4cc21e38c091c6d859202b54b
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11.6 MB | Preview Download |
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md5:70a1e1fd6cdcb6d998272c46b7e5e66c
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11.1 MB | Preview Download |
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md5:8daba0ebc9b980404716903474154ebf
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9.6 MB | Preview Download |
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md5:245e267c7e21cf51c57dba9ab245c6c1
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11.7 MB | Preview Download |
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md5:45d3edfa5eb83a7023dee37f2d20a8fb
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15.5 MB | Preview Download |
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md5:62e3248d5a0d1303a6f2e9f824a4d862
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11.9 MB | Preview Download |
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md5:caf3f21dc9b7ba556c3afc1b4304b83e
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12.5 MB | Preview Download |
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md5:961e1a00fe085404126f1d2a69d2780e
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11.3 MB | Preview Download |
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md5:ba7fc71460ce4a7582d72e1c82bbbe67
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12.3 MB | Preview Download |
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md5:a8e8eda8962ce3d634e5e7dc695e0ccc
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10.8 MB | Preview Download |
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md5:c69d06a5834f9464001a875baa32eba6
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12.4 MB | Preview Download |
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md5:c39c884a57cb9c7b87cc5243d58de914
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9.9 MB | Preview Download |
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md5:628c0e8279c403585e099adf216b1d87
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11.0 MB | Preview Download |
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md5:870f9876661699cd25837df68feaaff2
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11.7 MB | Preview Download |
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md5:2bff937e34e91e8419c1802ae6437fa4
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11.8 MB | Preview Download |
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md5:630d4cf8447eaf35d179cc7594321a86
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13.8 MB | Preview Download |
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md5:0c2de80119c48713e43f2d66b51a6bdb
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10.8 MB | Preview Download |
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md5:8311467fc80e07229945f5aff7a467b8
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12.4 MB | Preview Download |
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md5:cc84eb1a06b9a2783f00ac19fc479704
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12.3 MB | Preview Download |
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md5:c2057ce476a7ffc252ed22d29769d8db
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12.4 MB | Preview Download |
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md5:2d12a00324672fea7663229b28a3bcd7
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11.7 MB | Preview Download |
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md5:8b1c0ac9923fa5c88939fc85929f927d
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9.6 MB | Preview Download |
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md5:3e7a1d66e35235975459ef274168f495
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10.4 MB | Preview Download |
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md5:71041a40cd2a2e5abce8fb6f403a1dce
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10.8 MB | Preview Download |
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md5:34d09ba0f0d88793d6ed2451226d37d7
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11.2 MB | Preview Download |
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md5:03a27c041ca1fa57b5e06d4be976b6c5
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11.3 MB | Preview Download |
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md5:19e42ea5b78f583718411614d63466a6
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13.8 MB | Preview Download |
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md5:971a3dcb9c3f4d6d331b9fbe427327ce
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10.0 MB | Preview Download |
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md5:ffdcacd133251a84261ad2de96fde859
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13.0 MB | Preview Download |
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md5:366dececfebe7b4939158a14d88336e7
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14.5 MB | Preview Download |
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md5:c2854502505a45ee83c58217ff9202c2
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14.3 MB | Preview Download |
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md5:e29fc73336d3cc645a74c2197377a0ee
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11.4 MB | Preview Download |
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md5:e3fa371a37953cd459133b16e9e29544
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14.7 MB | Preview Download |
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md5:d2c82ae6bfefca9032c40ce6302ec592
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11.2 MB | Preview Download |
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md5:2a94274072539838e6f1e0c2570a90e6
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12.7 MB | Preview Download |
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md5:ef09f476ef1ab3614a328472d5335a07
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16.7 MB | Preview Download |
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md5:92bd6f226a08aec0c4c7532bdbb7489a
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12.9 MB | Preview Download |
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md5:b6a676a7d3709055b845f7cc668d3b1b
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9.6 MB | Preview Download |
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md5:d941dc302c398fb33cc8e769db8a706f
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13.2 MB | Preview Download |
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md5:bf5b0dabcc96ed22e95454adf87717ea
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11.3 MB | Preview Download |
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md5:16a878ced0091f9db694ea5fb1e67a1e
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10.9 MB | Preview Download |
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md5:dfe2103d4b23f65582528c5e9b5e4690
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11.1 MB | Preview Download |
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md5:51049bfb307b27d59d6dde895a2c8623
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9.5 MB | Preview Download |
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md5:e0ed662ad4c5541466e27dcb123a914e
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17.2 kB | Download |
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md5:d46a3f0d96c1393caaed93225c7d6a4b
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16.6 kB | Download |
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md5:45d8d25ed8e342e87bbac53965db58bd
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6.6 MB | Download |
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md5:fcb1f0ddd7e46573b71c820bb5b29cba
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11.6 kB | Download |
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md5:461cdc4ed3daf88517691ec5ba2e540f
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14.8 kB | Download |
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md5:729624663a93f384d8ab7b26991a0bea
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15.1 kB | Download |