Retromer-deficient VPS35 mutants associate with N-Ras. HEK293 cells were transiently transfected with plasmids directing expression of the indicated proteins. Whole-cell lysates (input) were immunoprecipitated by anti-HA antibody (HA immunoprecipitation [IP]; A) or anti-FLAG antibody (FLAG immunoprecipitation; B), and samples were analyzed by immunoblot. Data shown are representative of at least three independent experiments.