The essential role of palmitoyl groups of H-Ras in RE targeting. (A) GFP–H-Ras was expressed overnight. Cells were treated with 25 µM 2BP in the presence of CHX for the indicated times followed by fixation to immunostain GM130. (B) Transferrin–Alexa Fluor 546 was endocytosed to COS-1 cells, which had been treated with 2BP for 16 h. Cells were fixed to immunostain GM130. (C–F) The indicated palmitoyl-deficient mutant proteins (C, GFP–H-Ras–C181-184S; D, GFP–H-Ras–C181S; E, GFP–H-Ras–C184S; F, GFP–H-Ras–C184L) were expressed overnight. Cells were treated with CHX for 4 h followed by fixation to immunostain GM130. The arrowhead indicates PM. Bars, 10 µm.