Fig. 5
From: Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation
Pathogenic LRRK2 causes kinase-dependent pericentrosomal/centrosomal accumulation of endogenous Rab8a. a, b Examples of non-transfected HEK293T cells (ctrl) or cells transfected with either wildtype or pathogenic LRRK2, or with kinase-dead pathogenic LRRK2 as indicated, and stained with γ-tubulin antibody, Rab8a antibody (rabbit polyclonal Rab8a antibody for panel a, sheep polyclonal antibody for panel b), and TOPRO. Scale bar, 5 μm. c Quantification of the percentage of cells displaying pericentrosomal Rab8a staining in either non-transfected cells (ctrl), or pathogenic LRRK2-transfected cells as indicated, either in the absence or presence of GSK2578215A (GSK) (500 nM, 1 h). Bars represent mean ± s.e.m., (n = 3 independent experiments); ****, p < 0.001; *, p < 0.05. d Cells were transfected with the indicated constructs, and extracts blotted for GFP-tagged LRRK2, endogenous Rab8a, and tubulin as loading control. e Cells were either left untreated or incubated with 500 nM GSK2578215A for 1 h as indicated, and extracts analyzed for phosphorylated (p-S935) or total (GFP) LRRK2. f Quantification of S935 dephosphorylation in cells expressing wildtype or mutant LRRK2 as indicated, in either the absence or presence of 500 nM GSK2578215A for 1 h. Bars represent mean ± s.e.m. (n = 3); ***, p < 0.005; **, p < 0.01