Fig. 5. Cx43 amino-acid substitution variants and Cx32 colocalize within connexin clusters at cell-cell appositions. Stably transfected BHK cells expressing Cx32 (BHK-Cx32) were co-transfected with wildtype or Cx43-GFP substitution variants, respectively. In all instances, both connexin isotypes localized to the same connexin clusters (labeled with arrows) as indicated by the yellow plaque color in the merged images (Merge). Cx32 was immunostained with Cx32-specific antibodies followed by TRITC-coupled secondary antibodies 24 hours after transfection and fixation (Anti-Cx32, red). Wild-type Cx43 and variants were detected by their GFP auto-fluorescence (GFP, green). Connexin clusters assembled between cells that expressed only Cx43 appeared green, clusters in cells that expressed only Cx32 appeared red (labeled with asterisks in the merged images of variants D12S, and R153W). Bar, 20 µm.