Fig. 3. Cys-less Cx43 structure, expression and function in Cx43-deficient granulosa cells. (A) Cx43 protein structure showing the six cysteine sites (red) on the extracellular loops that were mutated to form cys-less Cx43. (B) Schematic structure of the AP2 retrovector showing the segment encoding either cys-less Cx43 or wild-type Cx43 followed by an IRES-linked sequence encoding EGFP. The vector control contained only the IRES and the EGFP sequence. (C) Expression of wild-type (WT) Cx43 and cys-less Cx43 in Cx43-deficient granulosa cells. Infected cells were identified by EGFP expression (left column). Cx43 immunostaining showed mainly membrane expression of wild-type Cx43 with numerous gap junction-like plaques, and both membrane and cytoplasmic localization of cys-less Cx43. The overlay is shown in the right column. (D) Representative experiments showing the transfer of LY to neighboring cells after microinjection into one cell (asterisk) among the three treatment groups. Quantification of dye transfer (percentage of cells passing injected LY to more than one neighboring cell) is shown in the bar graph. The number of cells injected is shown above each bar. Scale bars, 10 µm.