Mouse anti gadph

The following antibodies were used for western blotting: rat anti-HA (3F10; Roche/Sigma-Aldrich), rabbit anti-myc, mouse anti-RhoA, rabbit anti-MLC2 (Santa Cruz Technology), rabbit anti-FAM40B (Sigma-Aldrich), rabbit anti-pThr18/pSer19-MLC2 (Cell Signaling), mouse anti-GADPH (Millipore), secondary HRP-conjugated sheep anti-mouse IgG and donkey anti-rabbit IgG (GE Healthcare). Antibodies for immunofluorescence analysis were: rabbit anti-HA (Santa Cruz Technology), rabbit anti-pSer19-MLC2 (Cell Signaling), mouse anti-VE-cadherin (BD Biosciences), rabbit anti-ZO-1 (#61–7300; ThermoFisher Scientific), secondary AlexaFluor-488 and -647-conjugated antibodies (Molecular Probes). Cells were also stained with DAPI (DNA) and AlexaFluor-546-labeled phalloidin (F-actin; Molecular Probes).

Proteins were separated by electrophoresis into pre-casted 4–20% SDS-PAGE gels (Biorad) and subsequently transferred onto Immobilon-P membrane (Millipore). Membranes were first incubated 1 h at RT with rabbit anti-LRRK2 phospho Ser935 (1:300, Abcam, RabMAbs cat#ab133450), rabbit anti-LRRK2 UDD3 (1:1000, Abcam, RabMAbs cat# ab133518) and mouse anti-GADPH (1:4000, Millipore), then with HRP-conjugated secondary antibodies (Sigma) for 1 h at room temperature and then incubated with enhanced chemiluminescent (ECL) western blot substrate (Thermo Scientific).