Figure 3.

In vitro interaction of Gα₁₂ mutants with LARG. Immunoblot results for all LARG binding-impaired Gα₁₂ cassette mutants and selected other mutants are shown. HEK293 cells were transfected with the indicated plasmids (7.0 μg per 10-cm plate) and lysates were prepared for co-precipitation assays as described in Methods. Prior to this step, 5% of each lysate was set aside as starting material (load). Pulldown experiments were performed on 7–9 mutants per experiment, plus myc-Gα₁₂^QL as a positive control, using equal amounts of GST-LARG-RH (LARG) immobilized on glutathione-sepharose. Immobilized GST was utilized in parallel as a negative control. For all experimental samples, 20% of the volume was analyzed by SDS-PAGE and Coomassie blue staining to verify equal amounts of GST-LARG-RH and GST proteins in the precipitates (data not shown). Immunoblots displayed in this figure are representative of at least three trials per cassette mutant, except for mutants A-D, F-H, V, and KKK that showed minimal impairment in LARG binding after two trials. (Inset) Coomassie blue analysis of GST-fusion constructs expressed in bacteria and immobilized on glutathione-sepharose: GST-LARG-RH (LARG), GST-p115-RH (p115), and GST alone. Molecular weight standards (in kDa) are indicated at right.