Figure 6.

Immunoblot analysis of PTX-insensitive G proteins and PGE₁-stimulated photolabeling of G protein α subunits in human sperm membranes. (A) Membrane proteins (50 μg per lane) of human sperm (lanes 1, 3, and 5) and of mouse L cells (lanes 2, 4, and 6) were resolved by 13% SDS/PAGE, blotted, and incubated with antisera recognizing α_q/11 (lanes 1 and 2), α₁₂ (lanes 3 and 4), and α₁₃ (lanes 5 and 6). Molecular masses (kDa) are indicated on the left. (B Left) Membranes (150 μg per tube) were photolabeled with [α-³²P]GTP azidoanilide at 4°C (lane 1) or 30°C (lanes 2 and 3) in the absence (lanes 1 and 2) or presence (lane 3) of 1 μM prostaglandin E₁ and immunoprecipitated with antiserum AS 370 (α_q/11). Immunoprecipitates were resolved by 10% SDS/PAGE, and labeled α subunits were visualized by autoradiography. One of seven independent experiments is shown. (B Right) The amount of immunoprecipitated α_q/11 was determined by immunoblotting with a biotinylated IgG fraction of AS 370 and peroxydase-conjugated streptavidin. Lanes 4, 5, and 6 correspond to lanes 1, 2, and 3, respectively.