D816V and V560G proliferation was dependent of 4E-BP1 expression. (A) Alexa 488–tagged siRNA was used to detect the transfection efficiency of both HMC-1 (D816V) and α-155 (V560G) by flow cytometer. Histogram shows the percentage of transfected cells by scramble, p70S6k, and 4E-BP1 siRNA. (B) Western blot analysis of HMC-1 (D816V) transfected with siRNA. Both proteins 4E-BP1 and p70S6k have been deleted on cells (left and right panel, respectively). Protein extracts were resolved by SDS-PAGE. Loading of protein was assessed using polyclonal antibody against actin. Data represent a typical experiment out of 3. (C) 4E-BP1 and p70S6k siRNA sensitivities of HMC-1 (D816V; □) and α-155 (V560G; ▦) were assessed after 48 hours of culture. Data are means ± standard error of 3 separate experiments, each plated in triplicate. These data are statistically significant (*P < .05, **P < .01, ***P < .001).