Figure 4

Phosphorylation of endogenous C3G upon overexpression of Hck and its localization to the Golgi. (A) Cos-1 cells were transfected with expression constructs as indicated and whole cell lysates used in western blotting for pY504-C3G. ECL was used for detection. Blots were reprobed to show expression of C3G and the kinases. (B) Endogenous pY504 C3G colocalizes with c-Src. Cos-1 cells were transfected with the c-Src GFP fusion protein vector and cells stained for pY504-C3G expression (Cy3). c-Src expression was visualized as GFP fluorescence. Images shown are optical sections taken using the apotome. (C) Endogenous pY504-C3G localizes to the Golgi. Cos-1 cells were transfected with Hck along with VSVG-GFP and stained for pY504 C3G. Cells were left untreated (control) or treated with nocodazole (Noc) prior to fixation as described in Methods. Panels show optical section for pY504 by Cy3 and the VSVG-GFP by GFP fluorescence.