μ2 dephosphorylation is required for efficient AP2 uncoating. (A) AAK1 is active in CCVs. CCVs were incubated in the presence of ATP for various times as indicated. Samples were centrifuged and pellets (containing CCVs) and supernatants were assayed for phosphorylated μ2 using phosphospecific antibodies. (B and C) HEK293T cells were either mock transfected or transfected with HA-μ2^wt, HA-μ2^T165A, or HA-μ2^T156D. After fixation, cells were colabeled with 10-nm gold–conjugated anti-TfnR antibodies (B3/25) and 5-nm gold–labeled anti-HA antibodies. (B) Representative example of a labeled coated pit showing the anti-HA antibodies labeling the inner surface of the plasma membrane (arrow) and the anti-TfnR antibodies (arrowhead) marking the extracellular leaflet. Bar, 50 nm. (C) Anti-TfnR gold particles were counted in HA-positive pits. More than 50 pits were counted for each condition. Results are expressed as the number of pits counted containing zero, one, two, or three gold particles. (D) Cells were transfected with either HA-μ2^wt or HA-μ2^T156D and incubated with Texas red Tfn for 5 min at 37°C. After fixation and acid stripping of the surface Tfn, cells were labeled with anti-HA antibodies (green). Bar, 7 μm. (E) Quantitation of the overlap of HA with Texas red Tfn. The degree of overlap between HA and Texas red Tfn was set at 1 in HA-μ2^wt cells. Results are expressed as the fold change in overlap compared with HA-μ2^wt cells and are from two experiments and are significant at P < 0.05 (*). (F) Western blot comparing the levels of μ2 and phospho-μ2 in mock-treated cells and those treated with siRNA against hRME-6. Histogram shows the quantification of the ratio of phospho-μ2/μ2 ± the range of two experiments. (G) Western blot comparing the levels of μ2 and phospho-μ2 in cells overexpressing HA–hRME-6^wt and HA–RME-6^F1487A compared with mock-transfected cells (Con). Histogram showing the quantification of the ratio of phospho-μ2/μ2 ± SEM from three experiments.