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Rab3A WDTAGQERYRTITTAYYRGAM Rab3B WDTAGQERYRTITTAYYRGAM Rab3C WDTAGQERYRTITTAYYRGAM Rab3D WDTAGQERYRTITTAYYRGAM Rab5A WDTAGQERYHSLAPMYYRGAQ Rab5B WDTAGQERYHSLAPMYYRGAQ Rab5C WDTAGQERYHSLAPMYYRGAQ Rab8A WDTAGQERFRTITTAYYRGAM Rab8B WDTAGQERFRTITTAYYRGAM Rab10 WDTAGQERFHTITTSYYRGAM Rab12 WDTAGQERFNSITSAYYRSAK Rab29 WDIAGQERFTSMTRLYYRDAS Rab35 WDTAGQERFRTITSTYYRGTH Rab43 WDTAGQERFRTITQSYYRSAN Figure 1
A SHEEP POLYCLONAL B RABBIT POLYCLONAL Rab29 Rab3A Rab3B Rab3C Rab3D Rab5A Rab5BRab5C(Rab7L1) Rab8ARab8B Rab10 Rab12 Rab35 Rab43 MLi-2 + - + - + - + - + - + - + - + - + - + - + - + - + - + - Rab3 Rab5 Rab29 (Rab7L1) Rab8 Rab10 Rab12 Rab35 Rab43 MLi-2 + - + - + - + - + - + - + - + - Abcam rabbit number Rab5 pser84 S942D Rab8 pthr72 S874D E8263 E8268 Rab8 pthr72 Rab10 pthr73 S873D E8256 Rab12 pser106 S876D Rab29 pthr71 S877D E8259 E8261 Rab10 pthr73 HA HA Rab29 Rab3A Rab3B Rab3C Rab3D Rab5A Rab5B Rab5CRab8A Rab8B Rab10 Rab12 (Rab7L1) Rab35 Rab43 MLi-2 + - + - + - + - + - + - + - + - + - + - + - + - + - + - Rab3 pthr86 SA082 Rab29 pser72 SA136 Rab35 pthr72 SA083 Rab43 pthr82 SA334 HA Figure 2
A RABBIT POLYCLONAL MEFs G2019S WT WT R1441C WT R1441G WT KO MLi2 - - + - - + - - + - - + - - + - - + - - - E8263 (rabbit polyclonal prab8) Rab8A total 250 kda 250 kda 50 kda E8261 (rabbit polyclonal prab10) MJFF-total Rab10 clone-1 LRRK2 total LRRK2 ps935 -tubulin B 5 E8263 (prab8) E8261 (prab10) Relative prab signal 4 3 2 1 0 G2019S G2019S + MLi-2 WT WT + MLi-2 WT WT + MLi-2 R1441C R1441C + MLi-2 WT WT + MLi-2 R1441G R1441G + MLi-2 WT LRRK2 KO C SHEEP POLYCLONAL IP: total Rab12 WT R1441G MLi-2 - + - + prab12 NS IB: Rab12 ps106 IB: Rab12 ps106 (High exposure) Rab12 IB: total Rab12 Figure 3
A Specificity panel HEK293 overexpressed LRRK2-phosphorylated Rab proteins MJFF number Abcam number Rab3A Rab8A Rab10 Rab35 Rab43 B A549 cell line WT Rab8A Rab10 KO KO MLi-2 - + - + - + C Recombinant in vitro phosphorylated Rab10 Phospho-Rab10 1000 300 100 30 10 3 pg D clone-1 Mouse embryonic fibroblasts LRRK2[R1441G] clone-2 clone-3 clone-4 clone-5 clone-6 clone-7 clone-8 108-10 83-4 136-1 108-1 22-5 233-3 250-7 279-2 279-6 MLi-2 - + - + - + - + - + - + - + - + - + clone-9 MJFF-pRAB10 clone-1 MJF-21-108-10 250 kda 150 kda 100 kda MJFF-pRAB10 clone-2 MJF-21-83-4 75 kda 50 kda MJFF-pRAB10 clone-3 MJF-21-136-1 37 kda MJFF-pRAB10 clone-4 MJF-21-108-1 MJFF-pRAB10 clone-5 MJF-21-22-5 E Mouse embryonic fibroblasts MJFF-pRAB10 clone-6 MJF-21-233-3 G2019S WT WT R1441C WT R1441G WT KO MLi-2 - - + - - + - - + - - + - - + - - + - - - MJFF-pRAB10 clone-7 MJF-21-250-7 MJFF-pRab10 clone-1 (MJF-21-108-10) MJFF-pRAB10 clone-8 MJF-21-279-2 Ei 6 MJFF-total Rab10 clone-1 MJFF-pRAB10 clone-9 MJF-21-279-6 HA Total Rab8A Total Rab10 Total Rab10 Relative prab10 signal 4 2 0 -Tubulin G2019S G2019S + MLi-2 WT WT + MLi-2 WT WT + MLi-2 R1441C R1441C + MLi-2 WT WT + MLi-2 R1441G R1441G + MLi-2 WT LRRK2 KO Figure 4
A clone-1 clone-2 clone-3 clone-4 clone-5 clone-6 clone-7 clone-8 clone-9 MJF-21-108-10 MJF-21-83-4 MJF-21-136-1 MJF-21-108-1 MJF-21-22-5 MJF-21-233-3 MJF-21-250-7 MJF-21-279-2 MJF-21-279-6 WT RG WT RG WT RG WT RG WT RG WT RG WT RG WT RG WT RG MLi-2 - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + - + 250 kda 150 kda 100 kda * NS 75 kda 50 kda SPLEEN 37 kda B 250 kda 150 kda 100 kda 75 kda 50 kda LUNG 37 kda C 250 kda 150 kda 100 kda 75 kda 50 kda KIDNEY 37 kda * NS D 250 kda 150 kda 100 kda * NS 75 kda 50 kda 37 kda BRAIN * NS E LUNG SPLEEN KIDNEY BRAIN WT RG WT RG WT RG WT RG MLi2 - + - + - + - + - + - + - + - + 250 kda 250 kda 50 kda IB: LRRK2 IB: LRRK2 ps935 IB: -Tubulin IB: Rab10 Figure 5
A MOUSE TISSUES (LRRK2 R1441C, 10 mg/kg MLi-2) LUNG SPLEEN KIDNEY BRAIN MLi-2 - - - - - + + + + + - - - - - + + + + + - - - - - + + + + + - - - + + + MOUSE 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 3 4 5 8 9 10 250 kda 150 kda 100 kda 75 kda 50 kda * NS clone-1 MJF-21-108-10 37 kda * NS * NS 250 kda 150 kda 100 kda 75 kda 50 kda clone-2 MJF-21-83-4 37 kda * NS 250 kda 250 kda MJFF-total Rab10 clone-1 LRRK2 total LRRK2 ps935 37 kda GAPDH B 100 MJFF-pRab10 clone-1 MJF-21-108-10 SIGNAL QUANTITATION 100 MJFF-pRab10 clone-2 MJF-21-83-4 SIGNAL QUANTITATION Relative prab10/gapdh signal 10 1 Relative prab10/gapdh signal 10 1 0.1 LUNG LUNG + MLi-2 SPLEEN SPLEEN + MLi-2 KIDNEY KIDNEY + MLi-2 BRAIN BRAIN + MLi-2 0.1 LUNG LUNG + MLi-2 SPLEEN SPLEEN + MLi-2 KIDNEY KIDNEY + MLi-2 BRAIN BRAIN + MLi-2 Figure 6
A Relative prab10 signal 2.5 2.0 1.5 1.0 0.5 0.0 Ctrl IPD * * * * * * Ctrl IPD Ctrl IPD 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 8 9 11 12 13 14 15 1612 13 14 15 16 1718 20 21 23 17 1819 20 21 22 23 24 24 25 26 27 28 29 30 25 MJFF-pRab10 clone-1 25 Rab10 25 Rab8a 250 37 LRRK2 p-erk1/2 37 ERK1/2 37 GAPDH Immunoblot B Ctrl IPD Ctrl IPD Ctrl IPD Ctrl IPD Ctrl IPD 1 2 3 4 5 1 2 3 4 5 6 6 7 8 9 11 7 8 9 10 1112 12 13 14 15 16 131415 16 17 18 17 18 19 20 20 2123 24 25 21 22 23 24 26 27 28 29 30 + Phospho-Rab 10 Total Rab 10 C 250 150 100 75 50 37 Ctrl IPD Ctrl IPD Ctrl IPD 1 2 1 2 1 2 1 2 1 2 1 2 Phos-tag immunoblot 25 20 83-4 108-10 136-1 MJFF-pRAB10 clone-2 MJFF-pRAB10 clone-1 MJFF-pRAB10 clone-3 25 37 Rab10 GAPDH Figure 7
A Specificity panel HEK293 overexpressed LRRK2-phosphorylated Rab proteins MJFF number Abcam number Rab3A Rab8A Rab10 Rab35 Rab43 B A549 cell line WT MLi-2 - + - + - + C Rab8A Rab10 KO KO Recombinant in vitro phosphorylated Rab8A Phospho-Rab8A 1000 pg 300 100 30 10 3 MJFF-pRAB8 clone-1 MJF-20-25-2 MJFF-pRAB8 clone-2 MJF-20-55-4 MJFF-pRAB8 clone-3 MJF-20-19-6 MJFF-pRAB8 clone-4 MJF-20-19-3 MJFF-pRAB8 clone-5 MJF-20-25-5 MJFF-pRAB8 clone-6 MJF-20-38-1 MJFF-pRAB8 clone-7 MJF-20-55-3 MJFF-pRAB8 clone-8 MJF-20-58-2 MJFF-pRAB8 clone-9 MJF-20-58-6 HA Total Rab8A Total Rab8A Total Rab10 -Tubulin Figure 8
Immunoprecipitation of endogenous Rab proteins MLi2 - + - + - + - + - + - + IP: MJFF-pRab8 clone-1 MJF-20-25-2 IP: MJFF-pRab8 clone-2 MJF-20-55-4 IP: Polyclonal prab8 E8263 WT RC WT RC WT RC IB: Rab8A IB: Rab10 IB: Rab35 Figure 9
A HEK293 HA-Rab8A overexpression 3000 3000 1000 300 100 30 10 MLi-2 + - - - - - - ng B HEK293 HA-Rab10 overexpression 3000 3000 1000 IB: MJFF-pRAB8 clone-1 (MJF-20-25-2) IB: Rab8A (CST) 300 100 30 10 MLi-2 + - - - - - - ng Phospho- Immunoblot * Phospho- Immunoblot * IB: MJFF-pRAB10 clone-1 (MJF-21-108-10) IB: Rab10 (CST) Phos-tag IB: MJFF-pRAB8 clone-1 (MJF-20-25-2) * * IB: Rab8A (CST) Phos-tag IB: MJFF-pRAB10 clone-1 (MJF-21-108-10) IB: Rab10 (CST) Rab8 Rab10 Figure 10
Supplementary Figure 1 ANTIBODY WB (lysates of overexpressing cells) BLEED 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th Rab3 pthr86 (SA082) Rab5 pser84 (S942D) Rab7L1 (Rab29) pthr71 (S877D) Rab7L1 (Rab29) pser72 (SA136) Rab8 pthr72 (S874D) Rab10 pthr73 (S873D) Rab12 pser106 (S876D) Rab35 pthr72 (SA083) Rab43 pthr82 (SA334 ) GOOD SIGNAL WEAK SIGNAL NO SIGNAL BLEED NOT AVAILABLE
Table 1. Antigens employed for sheep polyclonal phospho-specific and total Rab antibodies ANTIBODY SHEEP NUMBER ANTIGEN Rab3 pthr86 SA082 CAGQERYRT*ITTAYYR Rab5 pser84 S942D CAGQERYHS*LAPMYYR Rab7L1 (Rab29) pthr71 S877D CIAGQERFT*SMTRLYYR Rab7L1 (Rab29) pser72 SA136 CDIAGQERFTS*MTRLYYRSS Rab8 pthr72 S874D CAGQERFRT*ITTAYYR Rab10 pthr73 S873D CAGQERFHT*ITTSYYR Rab12 pser106 S876D CAGQERFNS*ITSAYYR Rab35 pthr72 SA083 CAGQERFRT*ITSTYYR Rab43 pthr82 SA334 CAGQERFRT*ITQSYYR Rab12 total Rab35 total Rab43 total SA227 SA314 SA135 Phosphorylated serine/threonine marked with S*/T* Rab12 (no epitope tag) (anity puried with 6HIS-SUMO-Rab12) GST-Rab35 (anity puried with MBP-Rab35) GST-Rab43 (anity puried with HIS-SUMO-Rab43)
Table 2. Antigens employed for rabbit polyclonal and monoclonal phospho-specific Rab antibodies ANTIBODY Rab8 pthr72 Rab10 pthr73 ABCAM project number MJF-20 MJF-21 ANTIGEN C-Ahx-AGQERFRT*ITTAYYR-amide Ac-AGQERFRT*ITTAYYR-Ahx-C C-Ahx-AGQERFHT*ITTSYYR-amide Ac-AGQERFHT*ITTSYYR-Ahx-C Phosphorylated serine/threonine marked with S*/T* RABBIT NUMBERS E8262- E8269 E8254- E8261 NAME OF FINAL MONOCLONAL ANTIBODIES MJFF-pRab8 MJFF-pRab10
Table 3. MJFF-pRab8 and MJFF-pRab10 monoclonal phospho-specific Rab antibodies MJFF CLONE NAME MJFF-pRab8 clone-1 MJFF-pRab8 clone-2 MJFF-pRab8 clone-3 MJFF-pRab8 clone-4 MJFF-pRab8 clone-5 MJFF-pRab8 clone-6 MJFF-pRab8 clone-7 MJFF-pRab8 clone-8 MJFF-pRab8 clone-9 MJFF-pRab10 clone-1 MJFF-pRab10 clone-2 MJFF-pRab10 clone-3 MJFF-pRab10 clone-4 MJFF-pRab10 clone-5 MJFF-pRab10 clone-6 MJFF-pRab10 clone-7 MJFF-pRab10 clone-8 MJFF-pRab10 clone-9 ABCAM CATALOG NUMBER MJF-20-25-2 MJF-20-55-4 MJF-20-19-6 MJF-20-19-3 MJF-20-25-5 MJF-20-38-1 MJF-20-55-3 MJF-20-58-2 MJF-20-58-6 MJF-21-108-10 MJF-21-83-4 MJF-21-136-1 MJF-21-108-1 MJF-21-22-5 MJF-21-233-3 MJF-21-250-7 MJF-21-279-2 MJF-21-279-6