Fig. 3. (  A ) Immunoblots of the heterotrimeric G-protein α subunits (Gα) Gα^q/11, Gα^s, Gαⁱ(isoforms 1–3), and Gα¹²control proteins (C) and the proteins present in the porcine tracheal smooth muscle crude membrane preparation (M). Total amounts of membrane protein loaded in the lanes were 25, 50, 75 and 100 μg for Gα^q/11, Gα^s, Gαⁱ, and Gα¹²immunoblots, respectively. (  B ) Effect of contractile agonists on the exchange of the nonhydrolyzable, radioactive form of guanosine 5′-triphosphate (GTP), [³⁵S]GTPγS, for guanosine 5′-diphosphate (GDP) ([³⁵S]GTPγS–GDP exchange) at Gα of heterotrimeric G proteins. [³⁵S]GTPγS–GDP exchange was measured in the absence (basal exchange) or presence of maximal stimulation (agonist-promoted exchange) with acetylcholine (100 μm), endothelin-1 (100 nm), or histamine (100 μm). Immunoprecipitation was performed using nonimmune serum (for background measurements), antiserum specific for Gα^q/11or Gαⁱ(isoforms 1-3), or immunoglobulin G antibody specific for Gα^sor Gα¹². Agonist effects on Gα¹²[³⁵S]GTPγS–GDP exchange were conducted only for endothelin-1, because functional coupling of this protein and muscarinic and histamine-1 receptors has not been demonstrated. Data are normalized to the total protein in the assay and are presented as mean ± SD; n=3. * Significant difference from background radioactivity. † Significant difference from basal Gα[³⁵S]GTPγS–GDP exchange. CPM=counts per minute.