Mjf r20

Both, LRRK2 auto-phosphorylation analyses of pS1292 and Rab8A phosphorylation assays were recently described in Weng et al.^{31 (link)}. Oxidation- and reduction-dependent phosphorylation of Rab8A was determined by adding 1 mM DTT, 250 µM H₂O₂, 250 µM NCA or 250 µM CXL-1020 to a kinase buffer (25 mM Tris pH 7.4, 50 mM NaCl, 10 mM MgCl₂, 500 µM GDP, 1 mM DTT, 0.1 mg/mL bovine serum albumin (BSA)), supplemented with 1 mM ATP, 2.5 µM His₆-Rab8A (6–175). Recombinantly expressed His₆-Rab8A (6–175) was purified from E.coli BL21(DE3) RIL cells using Ni²⁺-NTA agarose (MACHEREY-NAGEL). For detection of Rab8A phosphorylation a primary antibody against pT72-Rab8A (1:5000; Abcam MJF-R20) was used as well as an anti-His6-Rab8A antibody (1:1.000; Abcam ab18184). LRRK2 auto-phosphorylation on S1292 was detected with an anti-pS1292 (1:1000; Abcam MJFR-19–7–8) antibody, in addition to an anti-Flag antibody (1:1000, Sigma-Aldrich (F1804)). As secondary antibodies the RDye® 800CW Donkey anti-Rabbit IgG Secondary Antibody (1:15000; LiCOR 926–32213) and IRDye® 680RD Goat anti-Mouse IgG Secondary Antibody (1:15000; LiCOR 926–68070) were used. Detection and quantification were performed using an Odysseys FC imaging system (LiCOR). All blots or gels derive from the same experiment and they were processed in parallel.

Antibodies used in this study were diluted in 5% w/v bovine serum albumin in TBS supplemented with 0.1% Tween-20 (TBS-T) and 0.03% w/v sodium azide. The rabbit monoclonal antibody for total LRRK2 (N-terminus) was purified at the University of Dundee (16 (link)). Anti-GFP (PABG1; Chromotek, used at 1:1000), anti-HA (3F10; Merck, used at 1:1000), anti-pT72-Rab8a (MJF-R20; Abcam, used at 0.5 μg/mL), anti-LRRK2 C-terminal (N241A/34; Neuromab, used at 1:1000), and anti-α-tubulin (3873S; CST, used at 1:5000). Secondary antibodies used were LI-COR IRDye for 800CW goat anti-rabbit (925–32211), goat anti-mouse (926–32210), and 680LT goat anti-rat (925–68029) and goat anti-mouse (926–68020), all used at 1:10,000 dilution in TBS-T.