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. 2014 Apr;85(4):586–597. doi: 10.1124/mol.113.088443

Fig. 7.

Fig. 7.

Identification of additional determinants of SRE activation in Gα12. (A) Schematic of Gα12/Concertina chimeras is shown. Chimeras were engineered as described in Materials and Methods, with the switch regions demarcated by the N-terminal boundary of the switch I region (Cys276 of Concertina, Ala202 of Gα12) and the C-terminal boundary of the switch III region (Arg342 of Concertina, Arg267 of Gα12). An activating Gln-to-Leu mutation was engineered in the switch II region of each construct. For chimera 4-sub, the indicated 45-residue Concertina region was introduced to chimera 4 in place of the indicated 42-residue Gα12 region. (B) Stimulation of SRE-mediated transcription in HEK293 cells (see Materials and Methods) by Gα12/Concertina chimeras 1–6 is shown. SRE activation is displayed as percent of the positive control myc-Gα12QL (12QL) in the same experiment. Values are mean ± range for three or more independent trials per chimera, with only the mean displayed for samples with values ≤3% of positive control. Immunoblot levels of GFP, coexpressed in the IRES vector harboring each chimera, are shown (lower panel). (C) Effects of C-terminal Concertina substitutions within chimera 4 and G12/13 proteins are shown. N-terminally myc-tagged chimera 4 and chimera 4-sub were tested for SRE activation, and mean ± range is shown for three independent experiments. Lysates were examined by immunoblot (IB) analysis using anti-myc antibody; results from a representative experiment are shown. SRE activation assays for mutants harboring the aforementioned 45-residue Concertina region at the C terminus of myc-Gα12QL (12QL-Δ45) and Gα13QL (13QL-Δ45) are presented in comparison with positive controls myc-Gα12QL and Gα13QL, respectively, and results are shown as mean ± range for three independent experiments. Comparative expression levels of the two Gα12 constructs, and the two Gα13 constructs, were determined by immunoblot analysis using anti-Gα12 (Santa Cruz Biotechnology) and anti-Gα13 (EMD Millipore) antibodies, and images from a representative experiment are shown.