Large clathrin structures are lost from fibroblasts upon AP2μ2 depletion. (A) Maximum intensity of AP2-TagGFP2 at endocytic site in hESCs, NPCs, and fibroblasts relative to hESCs (mean ± SEM). ***, P < 0.001, Mann-Whitney test (three to eight cells analyzed, 600–10,000 tracks analyzed). (B) Quantification of AP2μ2 expression levels in the three cell types. AP2μ2 levels were analyzed by immunoblot and normalized to the loading control (mean ± SD). **, P < 0.01, unpaired two-tailed t tests (three independent experiments). (C) Representative TIRFM images of CLTA-TagRFP-T (magenta) and DNM2-EGFP (green) in fibroblast transfected with control or AP2μ2 siRNA. Bar, 2 µm. (D) Mean fluorescence intensity profile for CLTA-TagRFP-T (magenta line for control cells and dark magenta dashed line for AP2μ2 siRNA) and DNM2-EGFP (light green line for control cells and dark green dashed line for AP2μ2 siRNA) in fibroblasts transfected with control or AP2μ2 siRNA (four independent experiments, three to four cells analyzed per experiment, 1,000–2,600 tracks analyzed) Western blot analysis of cell lysates immunoblotted for AP2μ2 and GAPDH loading control. (E) Endocytic productivity in fibroblasts transfected with control or AP2μ2 siRNA (mean ± SEM). **, P < 0.01, one-way ANOVA and Dunnett’s test versus control (four independent experiments, three to four cells analyzed per experiment, 1,000–2,600 tracks analyzed). ESC, embryonic stem cell.