Rab5 regulates AP2 uncoating via modulation of PtdIns(4,5)P₂ levels. (A) Immunofluorescence images of HEK293T cells labeled with the anti-PtdIns(4,5)P₂ antibody 2C11 (left) or with 2C11 in the presence of excess GST-2xFYVE (middle) or GST pleckstrin homology domain of PLCδ (right). Bar, 7 μm. (B) Representative image of HEK293T cells transfected with rab5^wt or rab5^S34N, which were incubated with Cy5-Tfn, acid-stripped, and then stained for PtdIns(4,5)P₂ using mAb 2C11. The arrowheads indicate an overlap between the two shown enlarged in the inset. Bar, 9 μm. (C) Quantitation of the overlap between PtdIns(4,5)P₂ and Cy5-Tfn in HEK293T cells overexpressing rab5^wt or rab5^S34N. The degree of overlap between 2C11 and Cy5-Tfn was set at 1 in rab5^wt-transfected cells. Results are expressed as the fold change in overlap compared with rab5^wt-transfected cells and are the mean ± of three experiments where at least 25 cells were counted for each condition and are significant at P < 0.01 (**). (D) Representative images of siRNA-treated cells incubated with Cy5-Tfn and, after acid stripping, stained using 2C11. The arrowheads indicate an overlap between the two shown enlarged in the inset. Bar, 10 μm. (E) Quantitation of the degree of overlap between PtdIns(4,5)P₂ and Cy5-Tfn in cells treated with control siRNA and siRNA against hRME-6 and rabex-5. The degree of overlap between 2C11 and Cy5-Tfn was set at 1 in control-treated cells and the results are expressed as the fold change in overlap compared with control and are the mean of three experiments ± SEM where at least 25 cells were analyzed. Values are significant at P < 0.01 (**) for control versus hRME-6 knockdown and P < 0.05 (*) for rabex 5 versus hRME-6 knockdown.