Fig. 4. Intracellular Cx43/ß-gal is Triton X-100 soluble. 43ß2 cells (A-D) or control cells (E,F) were incubated with PBS alone (A,C,E) or PBS containing 1% Triton X-100 (B,D,F) for 30 minutes at 15°C, then washed, fixed and immunostained using mouse anti-ß-gal antisera (A,B) and rabbit anti-Cx43 (C,D) or rabbit anti-Cx43 alone (E,F). The cells were then stained with FITC-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG. ß-gal images were obtained at 350 millisecond exposure with gain settings of 15 for unextracted cells (A) and 100 for Triton X-100 extracted cells (B), while Cx43 images were obtained at 1.5 second exposure with gain settings of 70 (C,E) and 200 (D,F). Triton X-100 extracted nearly all intracellular Cx43/ß-gal (arrowheads), revealing the Triton X-100 resistant pool of Cx43/ß-gal at the plasma membrane (arrows). Note the relatively higher Cx43 immunofluorescence signal from Triton X-100 extracted control cells (F), as compared to 43ß2 cells (D). Bar, 30 µm. (G-I) Control (G), 43ß1 (H) and 43ß2 (I) cells were processed for EM immunogold labeling as described in Materials and Methods and labeled using mouse anti-Cx43/15 nm gold-conjugated goat anti mouse IgG (arrows) and rabbit anti-ß-gal/5 nm gold-conjugated goat anti-rabbit IgG (arrowheads). Labeling for both Cx43 and Cx43/ß-gal was present in 43ß1 and 43ß2 cells (H,I), but not control cells (G). Bar, 100 nm.
