Wbox2 interferes with AP2–clathrin interactions and alters CCP dynamics. Dual-color TIRFM imaging of ARPE cells expressing mRuby-CLCa and α-eGFP-AP2 and treated with Wbox2. (A and B) Single frame from TIRFM video (7.5 min/video, see Video 6) and corresponding kymographs from region indicated by gray line. (C) Background AP2 fluorescence intensity of cells incubated without or with Wbox2 (20 µM). (D and E) Dual-color cmeAnalysis with either AP2 (D) or CLC (E) assigned as primary channel showing percentage of α-eGFP-AP2 tracks labeled with mRuby-CLCa (D) or mRuby-CLCa tracks labeled with α-eGFP-AP2 (E) determined in the presence of increasing concentrations of Wbox2. Data presented were obtained from a single experiment that is representative of two independent repeats. Number of videos acquired and analyzed: 11 for 0 µM, 10 for 5 µM, 10 for 10 µM, and 11 for 20 µM. Number of dynamic tracks analyzed in D: 43,324 for 0 µM, 34,664 for 5 µM, 33,596 for 10 µM, and 49,273 for 20 µM. Number of dynamic tracks analyzed in E: 65,192 for 0 µM, 42,323 for 5 µM, 49,733 for 10 µM, and 57,758 for 20 µM. Error bars are SD. ***, P ≤ 0.001.