Fig. 2. RIC-8 is required for interaction between GPA-16 and GPR-1/2, and for normal GPA-16 protein levels. (A) Embryonic extracts of the indicated genotypes were immunoprecipitated with GPA-16 antibodies and analyzed by western blot using RIC-8, GPR-1/2 or GPA-16 antibodies, as indicated on the left by arrowheads. The top panel shows inputs (1/50 of starting materials). Whereas GPA-16 antibodies co-immunoprecipitate both RIC-8 and GPR-1/2, RIC-8 or GPR-1/2 antibodies do not co-immunoprecipitate GPA-16 (data not shown). Lanes 1-5 and 7-11 are from the same experiment; lanes 6 and 12 from a different experiment performed with appropriate controls. GPA-16 levels are diminished in inputs from the gpb-1(RNAi) embryonic extract. Quantifications of the intensity of the GPA-16 band from four experiments, using the -tubulin or the GPR-1/2 band as loading control, indicate that the amount of GPA-16 in ric-8(md1909) and in gpb-1(RNAi) embryos is essentially identical [ratio of gpb-1(RNAi) versus ric-8(md1909):1.15; s.d.=0.4]. RIC-8 is truncated in ric-8(md1909) mutant embryos (Afshar et al., 2004) and is present at a lesser abundance than in the wild type. (B) Western blot analysis of embryonic extracts of the indicated genotypes using sequentially antibodies against GPA-16, GOA-1 and RIC-8, as well as -tubulin as a loading control, as indicated on the left with arrowheads. RIC-8 antibodies also detect a minor non-specific species, which co-migrate with the truncated protein in ric-8(md1909) mutant embryos (Afshar et al., 2004).