Fig. 2

a and b Establish VPS50 knockout cell line. The immunofluorescence and western blotting showed that the VPS50 was totally knocked out in Hela cells. c and d p. Gly169Val mutation delayed Transferrin recycling. Transferrin was lost more rapidly in Hela cells than in VPS50-KO cells and the p. Gly169Val mutation followed the same pattern of delaying as VPS50-KO cells. 4μg wild-type or different VPS50 mutations were transfected into VPS50-KO cells. After 24 h, puromycin was added into the DMEM for 24 h to filter untransfected cells. Transferrin chase was carried out using a modification of a previously described protocol. Inset bar, 20um (∗∗P < 0.01, two-tailed t-test)