Ríos-Cardona D , Ricardo-González RR , Chawla A , Ferrell JE .

GM46383 NIGMS NIH HHS , R01 GM046383-15 NIGMS NIH HHS , R01 GM046383-16 NIGMS NIH HHS , R01 GM046383-17 NIGMS NIH HHS , R01 GM046383-17S1 NIGMS NIH HHS , R01 GM046383-18 NIGMS NIH HHS , R01 GM046383 NIGMS NIH HHS , T32 AI007290 NIAID NIH HHS

Fig. 3. Characterization of the GPRx protein. (A) In vitro transcription and translation of GPRx in rabbit reticulocyte lysates. The GPRx cDNA (1 μg per reaction) was transcribed with SP6-RNA polymerase and translated in the presence of [35S]methionine. The reactions were analyzed by SDS PAGE followed by autoradiography and GPRx immunoblotting. (B) Ectopic expression of GPRx in Xenopus laevis oocytes. Oocytes were injected with GPRx mRNA (32 ng/oocyte) and allowed to express for 24 h. Membranes were prepared by centrifugation at 12,000 rpm and treated with or without endoglycosidase H (Endo H, 500 U per reaction) for 1 h. Proteins were analyzed by GPRx immunoblotting. (C) In vitro translation of GPRx in Xenopus laevis egg extracts. GPRx mRNA (1 μg) was translated in the presence of [35S]methionine. Samples of the whole reaction mixture (“Extract”) or membrane fractions (“Membrane”) were analyzed by autoradiography and GPRx immunoblotting. (D) Endogenous oocyte proteins recognized by the GPRx antibody. Membranes were prepared by 95,000 rpm centrifugation. Lane 1, membranes from stage VI oocytes; lane 2, membranes from stage VI oocytes treated with 5 μM progesterone to induce maturation; lane 3, membranes from stage VI oocytes injected with 64 ng morpholinos; lane 4, supernatant from stage VI oocytes; lane 5, supernatant from stage VI oocytes treated with 5 μM progesterone to induce maturation; lane 6, supernatant from stage VI oocytes injected with 64 ng morpholinos. The inset shows a section of the same blot with the contrast and brightness adjusted to make the faint p28 band more apparent. (E) Accumulation of the p50 protein during oogenesis. Membranes were prepared by 14,000 rpm centrifugation. From lane 1 to 6, all stages oocytes; lane 7, membranes from stage VI oocytes treated with 5 μM progesterone to induce maturation; lane 8, membranes from stage VI oocytes injected with 64 ng morpholinos. (F) cAMP levels in oocytes microinjected with GPRx mRNA (23 ng/oocyte), GPRx morpholino oligonucleotides 51 ng/oocyte), or control morpholinos (50 ng/oocyte). Data are shown as means ± S.E. from 20 experiments and are expressed relative to uninjected controls. The difference between the cAMP levels in the GPRx mRNA-injected oocytes and uninjected controls was statistically significant by the Student's t-test (P = 0.0009). The difference between the cAMP levels in the GPRx morpholino-injected oocytes and the control morpholino-injected oocytes was 3 ± 8.6%, which was not significant.

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