Membrane trafficking in AK1- and AK1B-transfected cells. (A) RPE-1 cells were transfected with VSVG-GFP (control) or cotransfected with VSVG-GFP and either flag-AK1 or flag-AK1B as indicated. VSVG-GFP was detected at the PM by incubating with the ectodomain antibody 8G5 during the last 30 min of a 60- or 100-min period after the shift at a permissive temperature. Cells were then fixed and stained as indicated (top images). 8G5 labeling was also quantified by using MetaMorph (n = 20 for each condition). Error bars indicate standard deviations. (B) Identical experiment as in A, but at different times after the permissive temperature shift, cells were fixed and stained as indicated. Arrows indicate the CTR, and arrowheads show the GA. (C, left) Video frames showing translocation of venus-NPY–containing vesicles in control cells, AK1-, or AK1B-expressing cells. (middle) Venus-NPY images recorded over 2 min were overlaid to visualize vesicle tracks. (right) Randomly selected particles were tracked for 2 min, and movement tracks are represented in different colors over a phase-contrast image of the recorded cell. Dashed lines indicate the cell border. Bars: (A and B) 10 µm; (C) 2.5 µm.