VPS35 is required for GTP loading of Ras and proliferation of N-Ras mutant melanoma cells. (A and B) HEK293 cells were transfected with untargeted siRNA (NT) or a validated pool targeting VPS35 or VPS26A. After 3 d, the cells were serum starved overnight and stimulated with EGF for the indicated time. The indicated proteins in cell lysates were quantified by immunoblot, and GTP loading was analyzed by GST-RBD pull down. Data shown are representative of four independent experiments. (C) Proliferation as measured by an MTS assay of SK-MEL-28 and -173 melanoma cell lines (BRAF or NRAS mutant, respectively) stably transfected with a tetracycline-inducible shRNA targeting NRAS or VPS35. shRNA was induced on the day of plating. Data shown are relative MTS values from day 1 to day 7 (top two panels) or at day 7 (bottom) after plating (mean ± SEM, n = 4; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant compared with untargeted siRNA). (D) Immunoblots show MEK, Erk, and Akt phosphorylation in the melanoma cell lines used in C.