Inhibition of CME prevents RA formation. (A) U2Os-AP2-GFP cells silenced for AP2A1, AP2S1, or control (scrambled shRNA) were plated on non-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (B) Analysis of RA coverage from samples in A. N (images): control = 12, shAP2A1#1 = 8, shAPA1#2 = 11, shAPS1#1 = 10, shAPS1#2 = 19, shAPS1#3 = 10. Data were obtained from two individual experiments and similar results were observed in five independent experiments. One-way ANOVA with Tukey’s multiple comparison, F(5, 64) = 43.11, P < 0.001. (C) U2Os-AP2-GFP cells overexpressing Ap180 ct were plated on non-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from samples in C; n = 17 from one representative experiment and similar results were observed in four individual experiments. Two-tailed Student’s t test, P < 0.0001. (E) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.