Silencing VPS35 promotes a vesicular distribution of GFP–N-Ras. (A and B) U2OS cells were transfected with untargeted siRNA (NT) or a validated pool targeting VPS35 or PDE6D. After 4 d, the cells were transfected with plasmids directing expression of the indicated fluorescent proteins and imaged live the next day. Representative images of U2OS cells expressing one of the four Ras isoforms (A) or coexpressing both N-Ras and β-1,4-galactosyltransferase (GalT; B). Bars, 10 µm.