Skip to main content
. 2014 Oct 1;127(19):4292–4307. doi: 10.1242/jcs.154864

Fig. 2.

Fig. 2.

RCC2 inhibits GEF-mediated activation of Rac1. (A) Comparison of GTP hydrolysis by GTPases in the presence of GFP (negative control), p50RhoGAP (positive control) and GFP–RCC2, n = 4. The image is of a Coomassie-stained gel. (B) GFP–RCC2 from 293T lysates bound preferentially to GDP-loaded, rather than nucleotide-free or GTPγS-loaded GST–Rac1, n = 7. *P<0.05 (ANOVA). (C,D) GFP–RCC2, but not the RCC2-K439E mutant, inhibited TrioD1-mediated loading of mant-GTP on to Rac1 (C), but had no effect on spontaneous GTP loading (D), n = 12 for each. (E) GFP–RCC2 failed to inhibit p50RhoGAP-catalyzed GTP hydrolysis by Rac1, n = 12. (F) GFP–Rac1 was precipitated by GFP-Trap from control, RCC2-knockdown and Coro1C-knockdown MEF lysates. Co-precipitated RhoGDI, RCC2 and Coro1C were detected by western blotting, n = 4. (G) GFP–RCC2 was precipitated by GFP-Trap and blotted for RhoGDI and RCC2. n = 4. For A and C–E, tagged RCC2 and TrioD1 proteins were purified from 293T cells by GFP-Trap and relative protein loading was demonstrated by Coomassie-stained gel or western blotting. Results are mean±s.e.m.