FIGURE 5.

Phosphorylation of Ser-395 by PKA increases Cx50 gap junction coupling but has no effect on Cx50 protein level. A, CEF cells expressing exogenous Cx50, Cx50(S395A), or Cx50(S387A,S395A) mutant in the absence or presence of PKA activator: 8-Br-cAMP (1 mm) or forskolin (10 μm) for 2 h before scrape loading dye transfer assay using LY (green) as a probe for gap junctional coupling and RD (red) as a tracer for originally dye-loaded cells (upper panel). Bar, 50 μm. Both forskolin and 8-Br-cAMP significantly increased Cx50 gap junction coupling, and this increase was significantly reduced in cells expressing Ser-395 mutant (lower panel; n = 6). B, CEF cells expressing Cx50 or the S395A mutant were detected by Western blotting (WB) using anti-FLAG antibody (1:1000 dilution) after being treated with 8-Br-cAMP (1 mm) or forskolin (10 μm) for 2 h. The membranes were stripped and reblotted with monoclonal antibody against the control protein, β-actin.