Figure 6.

Mitochondrial targeting of Gα12Q229L mutants and their interaction with Hsp90. A) COS-7 cells were transfected with either pcDNA3.1-Myc-His₆, or Gα12Q229L-Myc constructs as indicated. Cells were harvested 48 h after transfection and fractionated into mitochondrial pellet (P10) and supernatant (S10). Fractions were probed by Western blotting for the presence of Hsp90 and Gα12 (the latter detected using either c-Myc or Gα12 antibody; note that mutations in the R6A and A constructs are within the epitope of Gα12 antibody and are likely to affect its ability to recognize respective proteins). B) Mitochondrial fractions were solubilized in 0.5% Nonidet P-40; their amounts were adjusted to contain similar amounts of Myc-tagged Gα12; and immunoprecipitation was performed using c-Myc antibody. The presence of Myc-tagged Gα12 and Hsp90 in immunoprecipitates was detected using respective antibodies. Bottom panel shows quantification of the Western blotting data for Hsp90 binding, normalized to c-Myc signal in respective samples. ECL quantification data are means of two independent experiments; error bars indicate values obtained in each experiment.