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- Ἑκάβη Ελευθερόπουλος
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1 Biochemical Journal: this is an Accepted Manuscript, not the final Version of Record. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date version is available at Please cite using the DOI 1.142/BCJ21783 ACCEPTED MANUSCRIPT which Use of Archiving the open article access is published. articles is Archiving permitted of based non-open on the access terms articles of the specific is permitted Creative in accordance Commons Licence with the under Policy of Portland Press (
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29 Figure 1 LRRK2 RAB1 NEUTROPHILS Eosinophil Basophil NK cells NK (dim) NK (bright) DC s Dendritic (plasmacytoid) Dendritic (myeloid) CD8 (effector memory RA) CD8 (effector memory) CD8 (central memory) CD8 (naive) Tregs (memory) Tregs (naive) PBMC T cells Th2 Th17 Th1 CD4 (effector memory RA) CD4 (effector memory) CD4 (central memory) CD4 (naive) B cells B (plasma) B (memory) B (naive) monocytes Monocyte (non classical) Monocyte (intermediate) Monocyte (classical) 1x1 5 2x1 5 3x1 5 4x1 5 5x1 5 2x1 6 4x1 6 6x1 6 Protein copies per cell Protein copies per cell
30 Figure 2 A Donor Gender Male Female Male Male Female Female Male Male Female Male Male Female Viability 98.9% 99.6% 99.5% 99.2% 99.4% 99.5% 99.1% 98.8% 98.9% 99.2% 99.8% 99.7% Purity 98% 99.3% 97.2% 98.7% 99% 99.1% 99% 96.6% 99% 99.3% 99% 99.8% Total protein amount (mg/condition) B 2 Total LRRK2 Full Length (i) Donor MLi-2: LRRK2 (C-Terminus) LRRK2 (N-Terminus) LRRK2 ps GAPDH Total LRRK2/GAPDH (286kDa species) Donor: Total LRRK2/GAPDH (2kDa species) 2 1 Total LRRK2 2 kda species (ii) Donor: C LRRK2 ps935/total LRRK2 (286kDa species) 2 1 Phospho-Ser935 LRRK2 Full Length (iii) Donor: Donor MLi-2: Phospho-Rab1 vs GAPDH (ii) prab1 Rab1 GAPDH LRRK2 ps935/ Total LRRK2 (2kDa species) 2 1 Phospho-Ser935 LRRK2 2 kda species (iv) Donor: Total Rab1/GAPDH 2 1 Total Rab1 (i) Donor: Phospho-Rab1 vs Total Rab1 (iii) prab1/gapdh 2 1 prab1/total Rab Donor: Donor:
31 Figure 3 Control G219S Control G219S Donor 12 Donor 17 Donor 23 Donor 5 Donor 2 Donor 7 Donor 1 Donor 18 Donor 13 Donor 21 Donor 19 Donor 26 Donor 22 MLi-2: prab1 LRRK2 ps Rab1 LRRK2 37 Control Idiopathic Control Idiopathic Donor 14 Donor 16 Donor 6 Donor 24 Donor 15 Donor 3 Donor 9 Donor 11 Donor 1 Donor 4 Donor 2 Donor 8 Donor 25 MLi-2: GAPDH prab1 LRRK2 ps Rab1 LRRK2 37 GAPDH
32 Figure 4 A B C D Total LRRK2/GAPDH (Full length) Total LRRK2/GAPDH (2 kda species) LRRK2 ps935/total LRRK2 (Full length) Total Rab1/GAPDH Donor 13 Donor 17 Donor 5 Donor 13 Donor 11 Donor 17 Donor 21 Donor 5 Donor 13 Control G219S Idiopathic 2.5 Control Donor 17 Donor 5 Donor 12 G219S Idiopathic Control G219S Idiopathic Donor 14 Donor 21 Donor 1 Donor 11 Donor 6 Donor 23 Donor 12 Donor 16 Donor 18 Donor 9 Donor 22 Donor 1 Donor 7 Donor 26 Donor 2 Donor 19 Donor 14 Donor 21 Donor 1 Donor 5 Donor 6 Donor 12 Donor 9 Donor 23 Donor 16 Donor 18 Donor 22 Donor 26 Donor 1 Donor 21 Donor 14 Donor 1 Donor 16 Donor 23 Donor 13 Donor 6 Donor 9 Donor 11 Donor 18 Donor 22 Donor 1 Donor 23 Donor 1 Donor 11 Donor 9 Donor 17 Donor 18 Donor 6 Donor 12 Donor 14 Donor 16 Donor 22 Donor 26 Donor 19 Donor 1 Donor 7 Donor 2 Donor 8 Donor 4 Donor 2 Carrier Total LRRK2 Full Length Donor 25 Donor 8 Donor 2 Donor 3 Donor 15 Donor 4 Donor 24 Phospho-Ser935 LRRK2 Full Length Donor 7 Donor 19 Donor 2 Donor 26 Donor 25 Donor 2 Donor 3 Donor 4 Donor 8 Donor 15 Donor 24 Total Rab1 Donor 25 Donor 24 Donor 15 Donor 3 Total LRRK2/GAPDH (Full length) ps935 LRRK2/Total LRRK2 (Full length) Total Rab1/GAPDH Total LRRK2 2 kda species Donor 2 Donor 7 Donor 19 Donor 2 Donor 25 Donor 8 Donor 3 Donor 15 Donor 24 Donor 4 Total LRRK2/GAPDH (2 kda species) Control Control Control Control G219S G219S G219S G219S Idiopathic Idiopathic Idiopathic Idiopathic
33 Figure 5 A B C D prab1/gapdh prab1/total Rab1 prab1/gapdh prab1/total Rab1 5 Control G219S 4 Idiopathic Donor 13 Donor 11 Donor 21 3 Control G219S Idiopathic Donor 6 Donor 18 Donor 13 Donor 13 Donor 11 Donor 6 Donor 6 Donor 13 Donor 18 Control G219S Idiopathic Control G219S Idiopathic Donor 6 Donor 1 Donor 18 Donor 5 Donor 23 Donor 9 Donor 12 Donor 17 Donor 14 Donor 16 Donor 1 Donor 26 Donor 11 Donor 12 Donor 14 Donor 16 Donor 1 Donor 21 Donor 5 Donor 23 Donor 9 Donor 17 Donor 18 Donor 1 Donor 23 Donor 21 Donor 14 Donor 5 Donor 9 Donor 17 Donor 12 Donor 2 Donor 1 Donor 19 Donor 16 Donor 1 Total phospho-rab1 vs GAPDH Carrier Donor 19 Donor 7 Donor 2 Donor 22 Donor 24 Donor 8 Donor 2 Donor 15 Donor 4 Donor 3 Donor 25 prab1/gapdh prab1/total Rab1 prab1/gapdh prab1/total Rab1 2. ** Total phospho-rab1 vs Total Rab1 Donor 7 Donor 26 Donor 22 Donor 24 Donor 3 Donor 15 Donor 2 Donor 8 Donor 4 Donor 25 Donor 26 Donor 19 Donor 7 Donor 2 Donor 22 Donor 8 Donor 24 Donor 15 Donor 2 Donor 3 Donor 4 Donor Control Control LRRK2-dependent phospho-rab1 vs GAPDH Control LRRK2-dependent phospho-rab1 vs Total Rab1 Donor 11 Donor 14 Donor 16 Donor 12 Donor 1 Donor 23 Donor 17 Donor 9 Donor 5 Donor 21 Donor 2 Donor 1 Donor 7 Donor 19 Donor 26 Donor 22 Donor 3 Donor 15 Donor 24 Donor 2 Donor 8 Donor 4 Donor 25 Control ** G219S * G219S G219S ** G219S Idiopathic Idiopathic Idiopathic * Idiopathic
34 Figure 6 A Neutrophils PBMCs Donor MLi-2: prab LRRK2 ps935 Rab1 LRRK2 (C-Terminus) 37 GAPDH B Total LRRK2/GAPDH Donor: LRRK2 ps935/total LRRK Total LRRK2 Full Length (i) Neutrophils Phospho-Ser935 LRRK2 Full Length (iii) Donor: Neutrophils PBMCs PBMCs Total Rab1/GAPDH Total Rab1 (ii) Donor: prab1/total Rab1 Neutrophils PBMCs Phospho-Rab1 vs Total Rab1 (iv) Donor: Neutrophils PBMCs
35 Figure 7 A Conc. (nm): MLi PF prab1 LRRK2 ps935 Rab1 LRRK2 (C-Terminus) B (i) prab1/total Rab Conc. (nm): B (ii) ps935/total LRRK MLi-2 PF MLi-2 PF GAPDH. Conc. (nm): C D (i) Donor 1 Donor 2 MLi-2 (min): prab1 LRRK2 ps935 prab1/total Rab MLi-2 (min): Donor 1 Donor Rab1 LRRK2 (C-Terminus) GAPDH D (ii) ps935/total LRRK MLi-2 (min): Donor 1 Donor
36 Figure 8 A Donor 1 Donor 2 SDS: DIFP: MLi-2: prab1 B Fresh: C: C: + + MLi-2: prab1 25 LRRK2 ps LRRK2 ps Rab1 25 Rab1 25 LRRK2 (C-terminus) 25 LRRK2 (C-terminus) 37 GAPDH 37 GAPDH C Donor 1 Donor 2 Donor 1 Donor 2 DIFP (.5 mm): PMSF (2.5 mm): prab1 25 LRRK2 ps Rab1 25 LRRK2 (C-Terminus) 37 GAPDH
37 Figure 9 A Time (h): MLi-2: Donor 1 Donor 2 Donor prab1 25 LRRK2 ps Rab1 25 LRRK2 (C-Terminus) 37 GAPDH B prab1/total Rab Donor 1 prab1/total Rab Donor 2 prab1/total Rab Donor LRRK2 ps935/total LRRK LRRK2 ps935/total LRRK LRRK2 ps935/total LRRK Time (h):
38 Figure 1 Donor MLi-2: Low prab1 Rab1 Exposure Medium prab1 Rab1 High prab1 Rab1 Phos-tag
39 Supplementary Figure 1 Rab1 MJFF-total Rab1 clone-1 2 g 5 g WT KO WT KO Rab1 rabbit mab CST # g 5 g WT KO WT KO 25 kda 15 kda 1 kda 75 kda 5 kda 37 kda IB: Rab1 25 kda 2 kda 5 kda IB: Tubulin
40 Supplementary Figure 2 Donor 3 Gating around cells Live cell gating Granulocyte gating Side Scatter % DAPI % Side Scatter % 2.39 % Forward Scatter Forward Scatter FITC (CD66b)
41 Supplementary Figure 3 A Donor 1 Live cell gating B cell and T cell gating Monocyte and granulocyte gating DAPI Live Cells 99.9 % APC (CD19) B cells 3.71 % * T cells 59.9 % V5 (CD14) Monocytes 15.1 % Granulocytes 1.2 % Forward Scatter PE (CD3) FITC (CD66b) B * corresponds to the monocyte and granulocyte parent gate
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encouraged to use the Version of Record that, when published, will replace this version. The most /BCJ
Biochemical Journal: this is an Accepted Manuscript, not the final Version of Record. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date
encouraged to use the Version of Record that, when published, will replace this version. The most /BCJ BIOCHEMICAL JOURNAL
Biochemical Journal: this is an Accepted Manuscript, not the final Version of Record. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date
encouraged to use the Version of Record that, when published, will replace this version. The most /BCJ
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