Figure 6. Activation status of Gαi3... | Rockefeller University Press

Figure 6.

$Figure 6. Activation status of Gαi3 regulates its interaction with GIV. (A and B) GIV binds preferentially to the inactive (Gai3G203A) form of Gai3. (A, Top) In vitro binding assays were performed using GST-Gαi3 (wt or mutants) and either HeLa cell lysates (GIV (HeLa)) or in vitro–translated GIV (GIV (TnT)). Bead-bound fractions were separated by SDS-PAGE. Bound GIV was analyzed either by immunoblotting or autoradiography (TnT). (B, Top) In vitro binding assay performed as in A with GST-Gαi3wt after GDP loading in the absence or presence of AlF4−. (A and B, Bottom) Ponceau S-staining of PVDF membranes to show equal loading of GST proteins. (C) GIV preferentially binds to Gαi3. In vitro binding assays were performed as in Bwith GST-Gαi3 wt or Gαs in the presence of GDP ± AlF4− loading to compare the relative binding of Gαs and Gαi3 to GIV. Gβ-subunits were used as a positive control. Bound GIV from HeLa cell lysates was visualized by immunoblotting (GIV (Low exp), low exposure; GIV (High exp), high exposure) and autoradiography (TnT), respectively. (D) GIV interacts with the inactive Gαβγ heterotrimer in vivo. GIV was immunoprecipitated from rat brain lysate in the absence (lanes 1–3) or presence (lanes 4–6) of GDP + AlF4−, and immune complexes were probed for GIV and Gβ subunits using anti–pan Gβ.$

Activation status of Gαi3 regulates its interaction with GIV. (A and B) GIV binds preferentially to the inactive (Gai3G203A) form of Gai3. (A, Top) In vitro binding assays were performed using GST-Gαi3 (wt or mutants) and either HeLa cell lysates (GIV (HeLa)) or in vitro–translated GIV (GIV (TnT)). Bead-bound fractions were separated by SDS-PAGE. Bound GIV was analyzed either by immunoblotting or autoradiography (TnT). (B, Top) In vitro binding assay performed as in A with GST-Gαi3wt after GDP loading in the absence or presence of AlF₄⁻. (A and B, Bottom) Ponceau S-staining of PVDF membranes to show equal loading of GST proteins. (C) GIV preferentially binds to Gαi3. In vitro binding assays were performed as in Bwith GST-Gαi3 wt or Gαs in the presence of GDP ± AlF₄⁻ loading to compare the relative binding of Gαs and Gαi3 to GIV. Gβ-subunits were used as a positive control. Bound GIV from HeLa cell lysates was visualized by immunoblotting (GIV (Low exp), low exposure; GIV (High exp), high exposure) and autoradiography (TnT), respectively. (D) GIV interacts with the inactive Gαβγ heterotrimer in vivo. GIV was immunoprecipitated from rat brain lysate in the absence (lanes 1–3) or presence (lanes 4–6) of GDP + AlF₄⁻, and immune complexes were probed for GIV and Gβ subunits using anti–pan Gβ.