CLASP2 is required for the proper formation of AJs. (A) WT mKer were infected with lentiviruses expressing either CLASP2 shRNA (CLASP2 knockdown [KD]) or scramble shRNA (scramble). The levels of CLASP2 were analyzed by immunofluorescence after selection with G418. (B) Western blot (WB) showing the levels of CLASP2 after infection with the corresponding lentiviruses. (C) Western blot showing the total levels of ECad, p120, and α-catenin (αctn) in scramble and CLASP2 knockdown mKer at different time points of a calcium-switch time course experiment. (D) Scramble and CLASP2-deficient mKer were subjected to calcium switch for 30 min, 2 h, 6 h, and 12 h and immunostained for p120. (E) Quantification of p120 levels at the membrane in scramble and CLASP2-deficient mKer. Random individual plot profiles were generated at sites of cell–cell adhesion (10 per cell, 25–35 cells), and the maximum fluorescence intensity was quantified. Data are normalized to scramble control values after 30 min of calcium treatment and represented as means ± SEM; **, P < 0.003; ***, P < 0.0001, Mann–Whitney U test. (F) Scramble and CLASP2-deficient mKer treated with calcium for either 6 or 12 h and immunostained for ECad. (G) Scramble and CLASP2-deficient mKer immunostained for α-catenin after 6-h calcium switch. Insets are magnifications of the boxed regions. Bars: (A, main images, D, and F) 25 µm; (G, main images) 50 µm; (A and G, insets) 5 µm.