Figure 7

Activation of Ras by the constitutively active mutants of the TPR1-interacting Gα chimeras. (A) HEK 293 cells were transiently co-transfected with Ras, FLAG-TPR1 and the wild-type or constitutively active mutants of Gα₁₆, C174, N200, pcDNA1 (A), N102, N155, N188, C164 (B), zβ3, zβ2β3, N188-C164, or N200-C164 (C). Cell lysates were immunoprecipitated with GST-bound Ras binding domain of Raf-1 (Raf-1^RBD) agarose. The cell lysates and the eluted protein samples were subsequently immunoblotted against anti-Ras, anti-FLAG, and anti-Gα₁₆ or anti-Gα_z antibody. The result of the densitometric analysis is shown above the immunoblots and the numerical values represent relative intensities of Ras activity expressed as a ratio of the basal (B), Gα₁₆-mediated Ras activity (C) (set as 1.0). Data shown represent one of three sets of immunoblots; two other sets yielded similar results.