Generation of GαqQ209L in pcDNA3.1(+) (Life Technologies) from Gαqwt was by QuikChange site-directed PCR mutagenesis as previously described (14). The same procedure was used to generate mutant expression plasmids encoding GαqV182S, GαqV184M, GαqI190N, and GαqI190W [all hemagglutinin (HA)–tagged in pcDNA3.1(+)] using the primers as specified below: V182S, 5′-GTGCTTAGAAGTCGAGTCCCCACTACAGGGATC-3′ (forward) and 5′-GATCCCTGTAGTGGGGACTCGACTTCTAAGCAC-3′ (reverse); V184 M, 5′-GCTTAGAGTTCGAATGCCCACTACAGGGATC-3′ (forward) and 5′-GTAGTGGGCATTCGAACTCTAAGCACGTCTTGTTG-3′ (reverse); I190N, 5′-CAGGATCAACGAATACCCCTTTGACTTACAAAG-3′ (forward) and 5′-CAAAGGGGTATTCGTTGATCCCTGTAGTGGG-3′ (reverse); I190W, 5′-CAGGGATCTGGGAATACCCCTTTGACTTAC-3′ (forward) and 5′-GTAAGTCAAAGGGGTATTCCCAGATCCCTG-3′ (reverse). All mutant constructs were verified by sequencing. For transient expression of the pcDNA3.1(+)-based constructs in HEK293 cells, polyethylenimine (PEI) (Polysciences) was used as transfection vehicle with a DNA/PEI ratio of 1 μg of DNA/3 μl of PEI for 1.8 × 106 cells in one well of a six-well plate. This optimal DNA/PEI ratio (quantity and volume) was scaled according to the well numbers and their surface area.

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