Characterization of Novel Molecular Mechanisms Favoring Rac1 Membrane Translocation
Fig 6
WDR26 and Coro1A coordinately work in a Rho GDI-dependent Rac1 translocation step.
(A) COS1 cells transiently expressing either AU5-Rac1 or AU5-Rac1R66E (right, red signals) with indicated combinations of EGFPs (left, green signals) and Myc-Rho GDI proteins (bottom, blue signals) were fixed, stained with antibodies to the Myc epitope, and analyzed by confocal microscopy. Scale bar, 20 μm. (B) Quantification of the Rac1 translocation index obtained in experiments shown in A. ***, P ≤ 0.001 compared to cells expressing AU5-Rac1 alone (mock). (C) Extracts from non-stimulated and EGF-stimulated control and CORO1A-knockdown cells (CORO1A KD) expressing the indicated EGFPs (top) were immunoprecipitated with antibodies to Rho GDI and subjected to WB with antibodies to Rac1 (top panel). After stripping, the blot was incubated with antibodies to Rho GDI (second panel from top). Aliquots of the same extracts were analyzed by WB to detect the indicated phosphorylated and total proteins (five bottom panels). (D) Example of the abundance of endogenous WDR26 (top panel) upon transfection of COS1 cells with the indicated siRNAs (top). As loading control, we used the abundance of GADPH (bottom panel). (E) COS1 cells transfected with the indicated combinations of siRNAs (top) and EGFP-Rac1 (left, green signals) were serum starved, EGF stimulated, fixed, stained with rhodamine-labeled phalloidin (red color) and subjected to confocal microscopy. Scale bar, 20 μm. (F) Quantification of the Rac1 translocation index obtained in experiments shown in C. ***, P ≤ 0.001 compared to cells expressing the scrambled siRNA.