DPser2849 and PKC activity are required for proper DP border localization. (A, left) PKC inhibition impairs DP border localization and induces a filamentous pattern. SCC9 cells treated with 12.5 μM BIM or DMSO for 30 min and immunostained for endogenous DP. (A, right) BIM-treated A431 DP-GFP cells. (B) PKA inhibition does not affect DP border localization. SCC9 cells in low calcium treated with 10 μM H-89 or DMSO and switched to high calcium for 3 h, immunostained for endogenous DP. Quantitative analysis of border fluorescence intensity confirmed that DP localization was comparable in H-89–treated cells. (C) Mutation of Ser2849 results in IF alignment (Godsel et al., 2005). Fixed A431 cells expressing DP-GFP (a′) or DP^S2849G-GFP (b′) for comparison with D. (D, a′) Selected stills from Video 4 of A431 DP-GFP cells treated with 12.5 μM BIM. The drug was added at beginning of video and maintained throughout the 90-min time course. (D, b′) Video 5 of untreated A431 DP^S2849G-GFP. Arrows indicate areas where new cell–cell contacts occur. Note the variability in the DP^S2849G-GFP pattern ranging from particulate to continuous filaments (compare with C). (E) Impaired DP border localization during PKCα knockdown. Calcium switch of SCC9 cells infected with PKCα small hairpin RNA retrovirus or empty virus immunostained for DP. DP border fluorescence was measured, normalized to background, and plotted on the right. Immunoblot analysis of PKC-deficient cells for DP, PKP2, DSG2, PKCα, or GAPDH loading control. Error bars represent SEM. Bars, 10 μm.