Ab203181

Proximity ligation assays (PLAs) were performed essentially as described [37 (link)] using DuoLink® PLA Technology according to manufacturer's instructions (Sigma–Aldrich; Duolink® In Situ PLA probe anti-rabbit PLUS (DUO92002), Duolink® In Situ PLA probe anti-mouse MINUS (DUO92004), Duolink® In Situ Detection Reagents Red (DUO92008)). Briefly, cells on Cell-Tak-coated coverslips were fixed in 4% PFA/PBS as described above, followed by three washes in PBS. Coverslips were blocked in blocking solution followed by incubation with primary antibodies overnight at 4°C. Primary antibodies were rabbit polyclonal anti-phospho-S1292-LRRK2 (1 : 1000; Abcam, ab203181) and mouse monoclonal anti-LRRK2 antibody (1 : 1000; UC Davies/NIH Neuromab, clone 241A/34, 75–235). The proximity ligation signal was visible as individual dots, and analyzed by confocal microscopy as described above. The number of PLA positive dots/cell was quantified from around 300 cells per condition from maximal intensity projections using Leica Applied Systems (LAS AF6000) image acquisition software, and various control experiments included in each assay run.

SDS-PAGE gels were transferred onto nitrocellulose membranes using a Bio-Rad Trans-turbo blot system. Membranes were blocked with either 5% skim milk or 3% BSA in Tris-buffered saline with Tween-20 for 30 or 60 min at RT, respectively. Primary antibodies used diluted in blocking buffer were rabbit anti-GDI (Soldati et al., 1993 (link)) 1:1,000, mouse anti-Rab9 (Lombardi et al., 1993 (link)) 1:1,000, mouse anti-Myc (9E10 Hybridoma culture supernatant undiluted), 1.2 µg/ml rabbit anti-pT73Rab10 (230261; Abcam), rabbit anti-pT71 Rab29 (ab241062; Abcam)  1:500, rabbit anti-phospho-Ser1292 LRRK2 (ab203181; Abcam) 1:1,000, chicken anti-GFP (GFP-1010; Aves)  1:1,000, rabbit anti-LRRK2 (ab133518; Abcam), rabbit antitubulin (11224-1-AP; Proteintech) 1:2,000, and mouse anti-LAMP2 culture supernatant (H4B4; Developmental Studies Hybridoma Bank). Primary antibody incubations were either 1 h at RT or overnight at 4°C. LI-COR secondary antibodies diluted in blocking buffer used were 680 nm donkey anti-mouse (1:10,000), 800 nm donkey anti-mouse (1:5,000), 680 nm donkey anti-rabbit (1:10,000), 800 nm donkey anti-rabbit (1:5,000), 680 nm donkey anti-chicken (1:10,000), and 800 nm streptavidin (1:5,000). Secondary antibody incubations were for 1 h at RT. Blots were imaged using an Odyssey Infrared scanner (LI-COR) and quantified using ImageJ software.