HEK293T cells were transfected with plasmids encoding His6-Gβ1/His6-γ2 and either HA-Gαq or HA-GαqQ209L. On the next day, the cells were starved overnight. Forty-eight hours after transfection, the cells were lysed with 1.0 ml of ice-cold TBS-Triton [50 mM tris (pH 7.5), 150 mM NaCl, and 1% Triton X-100] containing 20 mM imidazole, 1 mM PMSF (phenylmethylsulfonyl fluoride), leupeptin (10 μg/ml), aprotinin (10 μg/ml), 10 mM β-glycerophosphate, 1 mM NaF, and 1 mM sodium orthovanadate. Lysates were transferred to 1.5-ml tubes and centrifuged at 4°C for 10 min at a relative centrifugal force of 15,682g. Pulldown assays to isolate His6-tagged Gβ1γ2 heterodimers were performed with TALON His-tag affinity beads (Clontech, catalog no. 8908-2). TALON beads are charged with cobalt, which is known to bind to His-tagged proteins with high specificity. Centrifuged lysates were transferred to 1.5-ml tubes and incubated with 30 μl of TALON beads in an ice bath with constant shaking for 30 min. The beads were then washed three times with lysis buffer, suspended in 1× Laemmli buffer containing β-mercaptoethanol, boiled for 5 min, and centrifuged at a relative centrifugal force of 15,682g for 5 min before being subjected to Western blotting analysis. For total cell lysates, a fraction of lysates was diluted with 4× Laemmli buffer containing β-mercaptoethanol, boiled for 5 min, and centrifuged at a relative centrifugal force of 15,682g for 5 min before Western blotting was performed. For immunoprecipitations, cell lysates obtained from HEK293T cells transfected with plasmids encoding either HA-Gαq or HA-GαqQ209L were transferred to 1.5-ml tubes and incubated with anti-HA monoclonal antibodies on a rocking platform overnight at 4°C. The next day, 30 μl of protein G Sepharose (Millipore, catalog no. 16-266) was added and the incubation was continued for 3 hours. Beads were washed three times with lysis buffer and subjected to Western blotting analysis or thin-layer chromatography.

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