Fig. 1.

Construction, expression, and solubilization of class-distinctive Gα₁₂ mutants. (A) Amino acid sequence of Gα₁₂ is shown. Each class-distinctive residue, indicated by an oval, was mutated to its nonclass-distinctive form (above right of ovals) in myc-tagged, constitutively active Gα₁₂. Shaded areas indicate the switch I, II, and III regions, and the dashed box indicates the site of the activating Q229L mutation. (B) The indicated mutant constructs were expressed in HEK293 cells, from which detergent-soluble extracts were subjected to immunoblot analysis as described in Materials and Methods. All mutants were single amino acid substitutions except the double-mutant Q232E/Q234K (QE/QK). Extracts from cells transfected with the Q229L variant of myc-tagged Gα₁₂ (12QL) and empty pcDNA3.1 (vector) were analyzed in parallel.