Fig. 6. Immunofluorescent imaging of Cx50-S50P in vitro and in vivo. (A,B) Transiently transfected HeLa cells expressing (A) Cx50-S50P or (B) wild-type Cx46 proteins were immunostained and examined by fluorescence microscopy. Merged images taken at x100 (A) and x60 (B,C,D) exhibit Cy2 (green) and/or Cy3 (red) staining of connexins and DAPI staining of cell nuclei (blue). The images showed that Cx50-S50P alone fails to correctly localize to the cell membrane, instead showing the accumulation of connexin subunits in subcellular compartments surrounding the nucleus (A). Wild-type Cx46 was efficiently translated and localized to the membrane specifically at areas of cell to cell apposition (B, arrow). (C,D) HeLa cells cotransfected with both Cx50-S50P (C) and wild-type Cx46 (D) showed that in the presence of Cx46, S50P subunits were colocalized to the membrane and form junctional plaques with at areas of cell-cell contact. (E,F) Fluorescence images of Cx50-S50P immunostaining in the bow region of homozygous mutant lenses (E) with Cx46 (Cx50^(S50P/S50P)–Cx46^(+/+)) and (F) without endogenous Cx46 (Cx50^(S50P/S50P)–Cx46^(–/–)). These lenses taken from mice on postnatal day 7 showed more punctate Cx50 staining (green) which extended deeper into the lens differentiating fibers in the presence of wild-type Cx46 proteins. Frozen Lens sections were co-stained with Rhodamine-phalloidin (red) and DAPI (blue). Bars, 5 µm (A-D), 40 µm (E-F).