FIGURE 3.

Expression analyses of mPanx1 mutants with modified intracellular cysteine residues in injected oocytes. A, glycosylated membrane proteins of cRNA-injected oocytes were labeled with biotinyl-ConA and then lysed (left panel). The lanes marked total display a fraction of the total lysates before the pulldown was performed. The lanes marked PD show the fractions that were obtained after a pulldown with streptavidin-agarose. As a negative control, a pulldown was performed on Panx1 WT (wt)-injected oocytes omitting the ConA labeling (ø). The Western blot analysis was performed using a Panx1 antibody. ni, not injected. Number of oocytes: not injected, 45; WT ø, 14; WT, 9; C136S, 15; C426S, 20. The right panes shows Western blot analysis performed with total lysates of noninjected (ni), wild type (wt), and C346S-injected oocytes. The lysates shown derived from n = 3 oocytes isolated 24 h after injection. B, cRNA-injected oocytes were fixed with 2% paraformaldehyde and frozen. 12-μm-thick cryostat sections were used for immunocytochemistry and confocal fluorescence microscopy. Scale bar, 20 μm. The C346S-expressing oocyte was taken at the same time point as the other mutants but treated with Cbx to extend oocyte viability.