Fig. 2. ( A ) Immunoblots of crude membranes prepared from COS-7 cells transfected with vector pcDNA3.1 only or cotransfected with the complementary DNA (cDNA) constructs encoding for the human M3muscarinic receptor and human Gαq(M3–Gαqcotransfected). Each lane was loaded with 10 μg membrane protein. ( B ) Representative tracings showing the effect of acetylcholine (ACh) on cytoplasmic Ca2+concentration ([Ca2+]c) in fura-2–loaded COS-7 transfected with vector pcDNA3.1 only or cotransfected with the cDNA constructs encoding for the human M3muscarinic receptor and human Gαq. ( C ) Effect of ACh on inositol triphosphate (IP3) levels in COS-7 cells expressing human Gαqonly, or human M3muscarinic receptor and human Gαq. ( D ) Effect of acetylcholine on the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate (GTP), [35S]GTPγS, for guanosine-5′-diphosphate (GDP) ([35S]GTPγS–GDP exchange) at the α subunit of the Gqheterotrimeric G protein (Gαq). Assays were performed using crude membranes prepared from COS-7 cells expressing human Gαqonly, or human M3muscarinic receptor and human Gαq. Measurements were made in the absence (constitutive exchange) and presence of 10 μm ACh. The immunoprecipitation step of the assays was performed using nonimmune serum (for nonspecific background measurements) or antiserum specific for Gαq. Data are mean ± SD; n = 4. * Significant difference from background radioactivity. † Significant difference from constitutive Gα[35S]GTPγS-GDP exchange. CPM = counts per minute.