Fig. 9. Inhibition of PKA in the absence of de novo protein synthesis. (A) Oocytes were injected with mRNAs for myr-HA-ß₂AR-C (S345A) or myr-HA-ß₂AR-C(S346A). Following 24 hours incubation, PKA phosphorylation of the two mutants was analyzed by 2D electrophoresis followed by anti-HA immunoblotting. Arrow, myr-HA-ß₂AR-C; asterisk, nonspecific protein. (B) Oocytes were injected with myr-HA-ß₂ARC/GRK2- (lane 1), myr-HA-ß₂AR-C/PKA^- (lane 2), myr-HA-ß₂AR-C/S346A (lane 3), myr-HA-ß₂AR-C/S345A (lane 4) or myr-HA-ß₂AR-C (lanes 5-10). After 24 hours of incubation, oocytes in lanes 1-4 were directly lysed and analyzed by SDS-PAGE followed by immunoblotting with anti-phospho-ß₂AR-C (S345 346) (upper panel) or anti-HA (lower panel). Oocytes in lane 6 were treated with progesterone for 1 hour. Oocytes in lane 7 were injected with PKAc (0.8 units) 1 hour prior to the progesterone treatment. Oocytes in lanes 9 and 10 were treated with 100 µM H89 for 1 or 2 hours respectively. Representative examples from three to five independent experiments are shown. (C) Oocytes were injected with mRNA for wild-type myr-HA-ß₂AR-C. After 24 hours of incubation, half of the oocytes were pre-incubated for 2 hours in OR2 containing 50 µg/ml cycloheximide. Both groups were then incubated with progesterone. At the indicated times following the addition of progesterone, oocytes were scored for GVBD and ten oocytes were randomly withdrawn for lysis. The extracts were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. Shown are representative examples of five independent experiments.