Fig. 3.
PAR1 and PAR2 form stable complexes with G protein heterotrimers. CoIP studies were performed as described but for these experiments; either Gαo or Gα11 was cotransfected with PAR1 or PAR2 and pulled down in the presence of GTPγS in the presence and absence of agonist. Here, we have also used a pan-Gβ antibody to detect the presence of endogenous Gβ in the receptor/Gα complex. Top, Western blot analysis of immunoprecipitated G proteins with corresponding G protein-specific antibodies. Bottom, Western blot analysis of cell lysates (input) with corresponding G protein-specific antibodies. Antibodies to Gαo and to Gα11 were mixed in one tube to blot the entire membrane at once. The same goat anti-rabbit secondary antibody was then used, and proteins were visualized using ECL. IB, immunoblot.