Lds sample buffer 4

Samples were supplemented with NuPage LDS sample buffer 4× (NP0008), boiled for 5 min at 95 °C and 50 µg of protein per sample were run on a Bio-Rad Criterion™ TGX™ polyacrylamide gel (Cat #5671095) at 200 V for 37 min. Gels were transferred to nitrocellulose (Bio-Rad, Cat #1704159) on a Bio-Rad Trans-Blot® Turbo™ transfer system at 20 V for 10 min. The nitrocellulose was blocked for 1 h in 50% TBS (20 mM Tris, 0.5 M NaCl, pH 7.5), 50% Odyssey blocking buffer (Li-Cor; 927–40,000) and incubated overnight with primary antibodies in 50% TBS-T (20 mM Tris, 0.5 M NaCl, pH 7.5, 0.1% Tween 20) and 50% Odyssey blocking buffer at 4 °C. Following 3 × 5 min washes with TBS-T, the nitrocellulose was incubated at RT with IR Dye 800CW Goat anti-rabbit IgG (#926-32211) or IR Dye 680RD Goat anti-mouse IgG (#926-68070) secondary antibodies for 1 h, washed 3 × 5 min and scanned on the Li-Cor platform. A list of antibodies can be found in Supplemental Table 4 and uncropped blots in Supplemental Fig. 5.

Samples were supplemented with NuPage LDS sample buffer 4× (NP0008), boiled for 5 min at 95 °C and 5 µl were run on a Bio-Rad Criterion™ TGX™ polyacrylamide gel (Cat #5671095) at 200 V for 37 min. Gels were transferred to nitrocellulose (Cat #1704159) on a Bio-Rad Trans-Blot^® Turbo™ transfer system at 20 V for 10 min. The nitrocellulose was blocked for 1 h in 50% TBS (20 mM Tris, 0.5 M NaCl, pH 7.5), 50% Odyssey blocking buffer (Li-Cor; 927–40,000) and incubated overnight with primary antibodies in 50% TBS-T (20 mM Tris, 0.5 M NaCl, pH 7.5, 0.1% Tween 20), 50% Odyssey blocking buffer at 4 °C. Following 3 × 5 min washes with TBS-T, the nitrocellulose was incubated at RT with secondary antibodies for 1 h, washed 3 × 5 min and scanned at the Li-Cor platform.
All primary antibodies were used at 1:2000 dilution: pS1292 LRRK2 (MJF-19-7-8; ab203181), total LRRK2 (ab133474), pS935 LRRK2 (ab133450), cyclophilin B (ab16045), PDI (Cell Signaling; 2446), MEK1/2 (Cell Signaling; 9122), and Tom20 (sc-11415). Secondary antibodies were used at 1:15,000 dilution: IRDye^® 800CW Goat anti-Rabbit IgG (LiCor: 926–32211). All blots presented in each figure panel were derived from the same experiment and were processed in parallel.