Figure 3.

Co-immunoprecipitation of mPRα coupled to G protein α subunits in spotted seatrout oocyte membranes with specific inhibitory G protein (G_{i 1,2}, G_o) antibodies followed by immunodetection of seatrout mPRα by Western blot analysis. Membranes (10 μg) were incubated for 45 min. with vehicle or 290 nM 20 β-S, solubilized and then incubated overnight with 5 μl of Gα_i1,2 or Gα_o antibody or nonimmune rabbit serum (niR) and 50 μl of Protein A/G Plus-agarose beads. Immunoprecipitated seatrout mPRα protein was detected using the monoclonal PR10-1 antibody described in Zhu et al. (2003). Representative immunoblot is shown. There was a strong and specific association of Gα_i1,2 with seatrout mPRα, whereas the mPRα protein was not detected in the Gα_o or nonimmune rabbit serum immunoprecipitated samples. The amount of seatrout mPRα associated with Gα_i1,2 was decreased after treatment with 20β-S (reproduced from Pace, 2005 [81] with permission).