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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
Figure 2 A Donor 1 2 3 4 5 6 7 8 9 1 11 12 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) 1.1.36.48.59.72.56.71.67.21.79.26 1.4 B 2 Total LRRK2 Full Length (i) Donor 1 2 3 4 5 6 7 8 9 1 11 12 MLi-2: - + - + - + - + - + - + - + - + - + - + - + - + 25 15 25 15 25 15 LRRK2 (C-Terminus) LRRK2 (N-Terminus) LRRK2 ps935 37 GAPDH Total LRRK2/GAPDH (286kDa species) 1 -+-+-+-+-+-+-+-+-+-+-+-+ Donor: 1 2 3 4 5 6 7 8 9 1 11 12 Total LRRK2/GAPDH (2kDa species) 2 1 Total LRRK2 2 kda species (ii) -+-+-+-+-+-+-+-+-+-+-+-+ Donor: 1 2 3 4 5 6 7 8 9 1 11 12 C LRRK2 ps935/total LRRK2 (286kDa species) 2 1 Phospho-Ser935 LRRK2 Full Length (iii) -+-+-+-+-+-+-+-+-+-+-+-+ Donor: 1 2 3 4 5 6 7 8 9 1 11 12 Donor 1 2 3 4 5 6 7 8 9 1 11 12 MLi-2: - + - + - + - + - + - + - + - + - + - + - + - + 25 25 37 3 Phospho-Rab1 vs GAPDH (ii) prab1 Rab1 GAPDH LRRK2 ps935/ Total LRRK2 (2kDa species) 2 1 Phospho-Ser935 LRRK2 2 kda species (iv) -+-+-+-+-+-+-+-+-+-+-+-+ Donor: 1 2 3 4 5 6 7 8 9 1 11 12 Total Rab1/GAPDH 2 1 Total Rab1 (i) -+-+-+-+-+-+-+-+-+-+-+-+ Donor: 1 2 3 4 5 6 7 8 9 1 11 12 3 Phospho-Rab1 vs Total Rab1 (iii) prab1/gapdh 2 1 prab1/total Rab1 2 1 -+-+-+-+-+-+-+-+-+-+-+-+ Donor: 1 2 3 4 5 6 7 8 9 1 11 12 -+-+-+-+-+-+-+-+-+-+-+-+ Donor: 1 2 3 4 5 6 7 8 9 1 11 12
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: - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + 25 25 prab1 LRRK2 ps935 25 25 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: - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + + 25 25 GAPDH prab1 LRRK2 ps935 25 25 Rab1 LRRK2 37 GAPDH
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 1 4 3 2 1 4 3 2 1 8 6 4 2 Donor 13 Donor 17 Donor 5 Donor 13 Donor 11 Donor 17 Donor 21 Donor 5 Donor 13 Control G219S Idiopathic 2.5 Control 2. 1.5 1..5. 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 2.5 2. 1.5 1..5. 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) 2. 1.5 1..5. 1.5 1..5. 2. 1.5 1..5. Control Control Control Control G219S G219S G219S G219S Idiopathic Idiopathic Idiopathic Idiopathic
Figure 5 A B C D prab1/gapdh prab1/total Rab1 prab1/gapdh prab1/total Rab1 5 Control G219S 4 Idiopathic 3 2 1 Donor 13 Donor 11 Donor 21 3 Control G219S Idiopathic 2 1 6 4 2 6 4 2 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. ** 1.5 1..5. 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 25 2. 1.5 1..5. 3 2 1 2.5 2. 1.5 1..5. 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
Figure 6 A Neutrophils PBMCs Donor 1 2 3 4 5 6 1 2 3 4 5 6 MLi-2: - + - + - + - + - + - + - + - + - + - + - + - + 25 prab1 25 15 25 25 15 LRRK2 ps935 Rab1 LRRK2 (C-Terminus) 37 GAPDH B Total LRRK2/GAPDH -+-+-+-+-+-+-+-+-+-+-+-+ Donor: 1 2 3 4 5 6 1 2 3 4 5 6 LRRK2 ps935/total LRRK2 3 2 1 2 1 2. 1.5 1..5. Total LRRK2 Full Length (i) Neutrophils Phospho-Ser935 LRRK2 Full Length (iii) -+-+-+-+-+-+-+-+-+-+-+-+ Donor: 1 2 3 4 5 6 1 2 3 4 5 6 Neutrophils PBMCs PBMCs Total Rab1/GAPDH 1.5 1..5. Total Rab1 (ii) -+-+-+-+-+-+-+-+-+-+-+-+ Donor: 1 2 3 4 5 6 1 2 3 4 5 6 prab1/total Rab1 Neutrophils PBMCs Phospho-Rab1 vs Total Rab1 (iv) 8 6 4 2 - + - + - + - + - + - + - + - + - + - + - + - + Donor: 1 2 3 4 5 6 1 2 3 4 5 6 Neutrophils PBMCs
Figure 7 A Conc. (nm): 25 25 25 25 MLi-2 3 1 3 1 3 PF-6447475 1 3 1 3 1 prab1 LRRK2 ps935 Rab1 LRRK2 (C-Terminus) B (i) prab1/total Rab1 1.5 1..5. Conc. (nm): B (ii) ps935/total LRRK2 1.5 1..5 MLi-2 PF-6447475 3 1 3 1 3 MLi-2 PF-6447475 1 3 1 3 1 37 GAPDH. Conc. (nm): 3 1 3 1 3 1 3 1 3 1 C D (i) Donor 1 Donor 2 MLi-2 (min): 1 3 5 1 3 5 25 25 prab1 LRRK2 ps935 prab1/total Rab1 1.5 1..5. MLi-2 (min): Donor 1 Donor 2 1 3 5 1 3 5 25 25 37 Rab1 LRRK2 (C-Terminus) GAPDH D (ii) ps935/total LRRK2 1.5 1..5. MLi-2 (min): Donor 1 Donor 2 1 3 5 1 3 5
Figure 8 A Donor 1 Donor 2 SDS: + + + + DIFP: + + + + MLi-2: - + - + - + - + - + - + 25 prab1 B Fresh: + + -2 C: + + -8 C: + + MLi-2: - + - + - + 25 prab1 25 LRRK2 ps935 25 LRRK2 ps935 25 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): + + + + 25 prab1 25 LRRK2 ps935 25 Rab1 25 LRRK2 (C-Terminus) 37 GAPDH
Figure 9 A Time (h): 2 4 8 12 24 MLi-2: - + - + - + - + - + Donor 1 Donor 2 Donor 3 2 4 8 12 24 - + - + - + - + - + 2 4 8 12 24 - + - + - + - + - + 25 prab1 25 LRRK2 ps935 25 Rab1 25 LRRK2 (C-Terminus) 37 GAPDH B prab1/total Rab1 1.5 1..5. Donor 1 prab1/total Rab1 - + - + - + - + - + 1.5 1..5. Donor 2 prab1/total Rab1 - + - + - + - + - + 1.5 1..5. Donor 3 - + - + - + - + - + LRRK2 ps935/total LRRK2 1.5 1..5. LRRK2 ps935/total LRRK2 - + - + - + - + - + 1.5 1..5. LRRK2 ps935/total LRRK2 - + - + - + - + - + 1.5 1..5. - + - + - + - + - + Time (h): 2 4 8 12 24 2 4 8 12 24 2 4 8 12 24
Figure 1 Donor 1 2 3 4 5 6 7 8 9 1 11 12 MLi-2: - + - + - + - + - + - + - + - + - + - + - + - + Low prab1 Rab1 Exposure Medium prab1 Rab1 High prab1 Rab1 Phos-tag
Supplementary Figure 1 Rab1 MJFF-total Rab1 clone-1 2 g 5 g WT KO WT KO Rab1 rabbit mab CST #8127 2 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
Supplementary Figure 2 Donor 3 Gating around cells Live cell gating Granulocyte gating Side Scatter 25 2 15 1 5 98.7 % 5 1 15 2 25 DAPI 1 5 1 4 1 3 1 2 99.5 % Side Scatter 25 2 15 1 5 5 1 15 2 25 97.2 % 2.39 % 1 2 1 3 1 4 1 5 Forward Scatter Forward Scatter FITC (CD66b)
Supplementary Figure 3 A Donor 1 Live cell gating B cell and T cell gating Monocyte and granulocyte gating DAPI 1 5 1 4 1 3 Live Cells 99.9 % APC (CD19) 1 5 1 4 1 3 B cells 3.71 % * T cells 59.9 % V5 (CD14) 1 5 1 4 1 3 Monocytes 15.1 % 1 2 1 2 1 2 Granulocytes 1.2 % 5 1 15 2 25 Forward Scatter 1 2 1 3 1 4 1 5 PE (CD3) 1 2 1 3 1 4 1 5 FITC (CD66b) B * corresponds to the monocyte and granulocyte parent gate