Figure 6: GM130 depletion increases Ras activity.

(a) Working model: GM130 acts as a suppressor of RasGRF activity, thereby modulating the activities of Ras and Cdc42. According to this model, the knockdown of GM130 results in increased Ras activation and decreased Cdc42 activation. (b) HepG2 cells were transfected with the indicated siRNAs. After 72 h, cells were lysed and Ras pull down was performed using GST-RBD. The eluate from the beads was subjected to SDS–polyacrylamide gel electrophoresis followed by immunoblotting with a pan-Ras antibody (Ras-GTP). Five per cent of the original lysate was subjected to SDS–polyacrylamide gel electrophoresis followed by immunoblotting against tubulin and GM130. Bar graph represents a quantification of the amount of active Ras (Ras-GTP) normalized to tubulin. 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; **P<0.01).