Figure 2.

Necdin interacts directly and preferentially with activated Gαo. (A) His-Gαo and GST-Necdin fusion proteins were purified from bacterial lysates. His-Gαo (600 nM) was incubated with 30 μM GDP andr AlF₄⁻ (mixture of 10 mM NaF and 30 μM AlCl₃), as indicated, for 1 h at 30°C. Purified GST-Necdin (200 nM) was added to the reaction mixture and incubated for an additional 20 min at 20°C. Glutathione-sepharose 4B beads were added to the final reaction mixtures, and the beads analyzed by immunoblot analysis using antibodies against Gαo and Necdin. Input was loaded with 10% of His-Gαo used for the assay. The numbers beside blot indicate size marker (kDa). (B) Lysates of 293T cells expressing plasmids either GαoWT (3 μg) or GαoQ205L (3 μg) together with FLAG-Necdin (10 μg) were incubated with anti-Gαo antibodies. Immunocomplexes were analyzed with antibodies against FLAG and Gαo. Input was loaded with 10% 293T cell extracts used for the immunoprecipitation assay.