FIGURE 3:

In S mutants that bind ZO-1 throughout GJ plaques, many more Cx43 polypeptides are bound to and coprecipitate with ZO-1 than for WT Cx43. (A, B) HeLa cells were transfected with Cx43 WT and S mutants and processed 24 h later for coimmunoprecipitation analyses. Whole-cell lysates were incubated with anti-ZO-1 antibodies and samples were tested for both ZO-1 and Cx43 after SDS–PAGE and Western blot analyses. Representative gels are shown in A and B, and quantitative analyses (as percentages) of at least three independent analyses, followed by one-way ANOVA with a Dunnett multiple comparison test (compared with the WT), are shown below. Note that S mutants S365A and E, S368A and E, and S373A all coprecipitated significantly more Cx43 than did WT, while the S373E mutant coprecipitated significantly less. Untransfected HeLa cells not endogenously expressing Cx43 were analyzed in parallel. (C) In parallel, Triton X-100 solubility assays according to Musil and Goodenough (1991) were performed in Cx43 WT and mutant expressing MDCK cells 24 h posttransfection. Cells were lysed in buffer containing 1% Triton X-100 and centrifuged at 10,000 × g to separate cell nuclei and mitochondria. Supernatants were then centrifuged at 100,000 × g to separate plasma membrane GJs (insoluble) from cytoplasmic Cx43 (soluble). After SDS–PAGE and Western blot, samples were analyzed for both Cx43 and ZO-1. Comparable results were obtained. (D) Triton X-100 solubility assays were carried out in parallel as a positive control in endogenously Cx43-expressing primary PAE cells.