Figure 4

EGF induces membrane translocation of Tks5 in response to EGF treatment. (A) COS7 cells were transiently transfected with V5-Tks5 and after serum-starvation cells were stimulated with EGF (G, H, I) or left untreated (A, B, C). V5-Tks5^R42A construct was also transiently expressed in the cells and after serum-starvation they were treated with EGF (J, K, L) or left untreated (D, E, F). Cells were then fixed and processed for immunofluorescence. Subcellular localization of Tks5 was detected using V5-specific monoclonal antibody (B, E, H, and K). To visualize membrane ruffles, cells were also stained with TRITC-phalloidin (A, D, G, and J). Arrows indicate Tks5 present at the plasma membrane. The scale bar represents 20 μm. (B) Quantitative analysis of Tks5 translocation upon EGF treatment. COS7 cells were transiently transfected with V5-Tks5 and V5-Tks5^R42A constructs and after serum-starvation cells were stimulated with EGF or left untreated. Prior to stimulation a part of the cells expressing V5-Tks5 were treated with the PI 3-kinase inhibitor LY294002 as indicated. The percentage of cells translocated to the plasma membrane under different conditions was quantified by an observer who was blinded to cell treatment status (n = 100 cells for each group per experiment). Error bars represent the Standard Error of the Mean, SEM. ***: p < 0.001, **: p < 0.005