Fig. 4.

Regulation of Rab GTP binding ability by LRRK2-induced phosphorylation. a, b GTP-binding assay for Rab1a, 3c, 8a, and 35. In a, HEK-293 cells were transfected with either wild-type (WT) or phosphomutants (TA or RD) of Rab1a, 3c, 8a, or 35. In b, HEK-293 cells were transfected with Rab1a, 3c, 8a, or 35 (all WTs) alone (depicted as “None” in each set) or together with WT or mutants (G2019S or D1994A) of LRRK2. In a and b, cell lysates were incubated with γ-amino-hexyl-GTP agarose and GTP-bound Rabs were subjected to SDS-PAGE for western blot analysis. Shown are representative immunoblot images (top) and quantification of GTP-binding Rab levels (bottom) from three independent experiments. Data are mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; one-way ANOVA followed by Dunnett’s multiple comparison post hoc test