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Fig. 8 | Molecular Neurodegeneration

Fig. 8

From: Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation

Fig. 8

Knockdown of Rab8a significantly reverses the centrosomal deficits mediated by pathogenic LRRK2. a Representative Western blot of extracts from control cells (ctrl), or cells transfected with wildtype (wt) or Y1699C-mutant LRRK2, along with either ctrl-siRNA or Rab8a-siRNA as indicated, and blotted against Rab8a or tubulin as loading control. b Quantification of the type of experiments depicted in a, with levels of Rab8a normalized to tubulin and to Rab8a levels in the presence of ctrl-siRNA. Bars represent mean ± s.e.m. (n = 3–5 independent experiments); * p < 0.05. c Example of cells co-transfected with GFP-tagged pathogenic LRRK2 and either ctrl-siRNA or Rab8a-siRNA as indicated, and stained with pericentrin antibody and DAPI. Scale bar, 10 μm. d Quantification of the split centrosome phenotype in control cells transfected with either ctrl-siRNA or Rab8a-siRNA, or in cells co-transfected with wildtype or mutant LRRK2 as indicated. Bars represent mean ± s.e.m. (n = 3–5 independent experiments); **** p < 0.001; * p < 0.05. e Western blot of cell extracts transfected with either ctrl-siRNA or Rab8a-siRNA as indicated, and four hours later cotransfected with mutant LRRK2 and mRFP-tagged Rab8a, Rab8a-T72A, or siRNA-resistant versions thereof (res), and blotted against Rab8a and GAPDH as loading control. f Quantification of the split centrosome phenotype in the presence of ctrl-siRNA or Rab8a-siRNA, and in the presence of pathogenic mutant LRRK2 and mRFP-tagged Rab8a, Rab8a-T72A or siRNA-resistant versions thereof (res) as indicated. Bars represent mean ± s.e.m. (n = 3 independent experiments); *** p < 0.005; ** p < 0.01. g Quantification of centrosome reorientation in cells stably expressing flag-tagged wildtype or G2019S-mutant LRRK2 together with RFP-tagged wildtype or phospho-deficient T72A Rab8a 4 h after generating the wound (t = 4 h). Random orientation is expected to be 25%. n > 50 cells were quantified for each condition in each experiment. Bars represent mean ± s.e.m. (n = 3 independent experiments); **, p < 0.01; *, p < 0.05

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