Figure 4.

Activation of serum response element mediated transcription by Gα12 mutants. (A) Luciferase reporter assay results of selected cassette mutants. HEK293 cells grown in 12-well plates were co-transfected with the plasmids SRE-L (0.2 μg) and pRL-TK (0.02 μg), plus 1.0 μg of the plasmid encoding each cassette mutant indicated on the X-axis. Firefly luciferase values were normalized for Renilla luciferase values within each sample, and values are presented as a percent of the value calculated for myc-Gα₁₂^QL (Y-axis) within the same experiment. Mutationally active (12QL) and inactive (G228A) samples were analyzed in parallel. Results shown are a representative of two experiments performed per Gα₁₂ variant. (B) Expression level of Gα₁₂ mutants. A sample of each lysate was set aside prior to luminometry and analyzed by SDS-PAGE and immunoblotting using anti-Gα₁₂ antibody (Santa Cruz Biotechnology). For all samples, densitometric intensity was determined as described in Methods, then divided by positive control myc-Gα₁₂^QL levels within the same experiment, and SRE-L/Renilla values were adjusted to reflect this normalization for protein levels.