FIGURE 5.
Endogenous Gαs and Gαq separate from their cognate receptors (β2R and M3R) after activation. Surface β2 or M3 receptors were pre-labeled with anti-HA antibodies (IgG1) as before. The cells were then incubated at 37 °C for 30 min with mouse anti-MHCI antibody (IgG2a) and isoproterenol 1 mm (A) or carbachol 1 mm (B). After fixation, the cells were labeled, in the presence of saponin, with rabbit anti-Gαs (A) or anti-Gαq (B) antibodies, followed by 488-conjugated goat anti rabbit antibody. Internalized MHCI and HA-tagged receptors were visualized with isotype-specific antibodies 594-GAM-IgG2a and 633-GAM-IgG1. Insets show enlarged views and colocalization of G proteins with MHCI on endosomes. Images shown are representative of experiments that were repeated three times.