Figure 5: The GM130–RasGRF complex is regulated by serum.

(a) MCF7 cells were grown to confluency. Then, cells were serum starved for 2 h followed by stimulation with 10% FCS for the indicated time points. Cell lysates were subjected to immunoprecipitation against GM130. The immunoprecipitate was subjected to SDS–polyacrylamide gel electrophoresis followed by immunoblotting against the indicated proteins. (b) HeLa cells were transfected with control or GM130 siRNA 72 h prior to the experiment. After 48 h, cells were transfected with the plasmid encoding for the Cdc42 Raichu probe. Cells were serum starved for 2 h followed by stimulation with 10% FCS for 0, 1 and 5 min and FRET was measured as indicated in ‘Methods’. Results are presented as mean±s.d. from three independent experiments. Asterisks indicate statistically significant differences tested using analysis of variance with Newman–Keuls multiple comparison test (*P<0.05). (c) HeLa cells were transfected with the indicated siRNA. After 48 h, cells were transfected with empty vector or with a plasmid encoding haemagglutinin (HA)-tagged RasGRF1. After 24 h, cells were serum starved for 2 h followed by stimulation with 10% FCS for the indicated time points. Cell lysates were subjected to immunoprecipitation against HA. The immunoprecipitate was subjected to SDS–polyacrylamide gel electrophoresis followed by immunoblotting against the indicated proteins.