Rab5 regulates AP2 uncoating in vivo. HEK293T cells, either mock transfected or transfected with rab5^S34N, were incubated with Texas red Tfn for 5 min at 37°C. After fixation, the surface Texas red Tfn was removed by acid stripping and AP2 was visualized using the mAb AP.6. (A) Representative images of mock-transfected cells or cells overexpressing rab5^S34N labeled with Texas red Tfn and AP.6. The arrowheads indicate an overlap between the two shown enlarged in the inset. Bars, 6 μm. (B) Quantification of the degree of overlap of Texas red Tfn with AP.6 in mock-transfected cells and those overexpressing rab5^S34N. The degree of overlap between the two markers was set at 1 in mock-transfected cells. Results are expressed as the fold change in overlap compared with mock-transfected cells and are the mean ± SEM of three experiments where at least 25 cells were analyzed for each condition in each experiment. Values are significantly different at P < 0.01 (**). (C) Electron micrograph of a clathrin-coated pit labeled with 10-nm gold–conjugated B3/25 antibodies recognizing the ectodomain of TfnR. Bar, 50 nm. (D) The number of gold-labeled particles per pit was counted in untransfected HEK293T cells or those transfected with rab5^wt, rab5^Q79L, and rab5^S34N. Results are expressed as particles per pit as a percentage of the total number of pits counted. 100 coated pits were counted in each sample. (E) Quantitation of the number of coated pits per profile in untransfected HEK293T cells and those transfected with rab5^wt, rab5^Q79L, and rab5^S34N. Results are expressed as a percentage of pit number per profile in untransfected cells. For each population, at least 60 cell profiles were counted. (F) Representative image of mock-transfected HEK293T cells and those transfected with rab5^S34N, which were incubated with Texas red Tfn, acid stripped, and then stained for clathrin using mAb X22. The arrowheads indicate an overlap between the two shown enlarged in the inset. Bars: (top) 7 μm; (bottom) 6 μm. (G) The degree of overlap of clathrin, measured by immunofluorescence using mAb X22, with Texas red Tfn was measured in mock-transfected HEK293T cells or those transfected with rab5^S34N. The degree of overlap between X22 and Texas red Tfn was set at 1 in mock-transfected cells and the results are expressed as the fold change in overlap compared with mock-transfected cells and are the mean ± SEM of two experiments where at least 25 cells were analyzed in each experiment.