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Fig. 1. Presence of Cx33 in the rat testis. (A) Anti-Cx33 western blot analysis detected a band migrating at 33 kDa in lysates of testis but not of brain, heart, uterus and ovary. The anti-Cx43 antibody detected the presence of different phosphorylated Cx43 isoforms in all tissues (left panel). The numbers on the right indicate molecular mass (kDa). Immunolocalization of Cx33 in seminiferous tubules (right panel). Note that the Cx33 signal is only present in the basal compartment of seminiferous tubule (x120) whereas Cx43 is detected in both tubular and interstitial compartments (inset). (B,C) Characterization of the unphosphorylated status of Cx33. (B) Testis immunoprecipitates (Ip) with anti-Cx33 or anti-Cx43 antibodies were treated with (+) or without (-) alkaline phosphatase (AP) and analyzed by western blotting with Cx33 or Cx43 antibodies (left panels). Testis immunoprecipitates with anti-Cx33 were analyzed by western blot with Cx33, phosphotyrosine (Ptyr), phosphoserine (Pser) or phosphothreonine (Pthr) antibodies (right panel). (C) COS-7 cells were transfected with Cx43, Cx33-myc or empty vector (-) and labeled with [³²P]orthophosphate. Cell lysates were then immunoprecipitated (Ip) with myc (left panel) or Cx43 (right panel) antibodies and separated by SDS-PAGE. The [³²P] radiolabeled proteins were visualized by autoradiography and the presence of Cx33-myc and Cx43 was verified by western blotting with myc or Cx43 antibodies. The positions of Cx33 and Cx43 isoforms are shown by arrowheads. Figures are representative of three separate experiments. Open arrowheads indicate positions of unphosphorylated and phosphorylated forms of Cx43.

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