Colocalization of clathrin-coated pits and 2031-ACP-IR upon insulin stimulation. (A) 2031-ACP-IR stable expressing cells transfected with AP2-mEGFP plasmid were serum-starved for 2 h and cells were labeled with CoA-Cy3, then time-lapse imaging was performed every 1 min after adding control solution for 3 min and 100 nM insulin solution for 3 min. Left; AP2-mEGFP, middle; 2031-ACP-IR labeled with CoA-Cy3, right; merge. Scale bar, 10 μm. (B) Examples of segmented images of AP2 (left) and ACP-IR (right) in cell1, cell2, and cell3 showed in A). (C–E) Segmented images were used for colocalization analysis in C, D, and E. Scale bar, 10 μm. Colocalization between AP2 clathrin adaptor and 2031-ACP-IR was analyzed by MCC (C and D) or SRCC (E). Data represent the mean ± SEM of six time-lapse data sets (n = 20 cells). Statistical significance was calculated by one-way ANOVA, ns, not significant. (C) The MCC estimated as the ratio of the AP2 colocalized with ACP-IR over the total fluorescent of AP2. (D) The MCC estimated as the ratio of the ACP-IR colocalized with AP2 over the total fluorescent of ACP-IR.