Figure 2.

siRNA-mediated depletion of PhLP inhibits Gβγ dimer assembly. (A) HEK-293 cells were treated with the indicated siRNA and transiently transfected with Gβ_l and HA-Gγ₂ and then assayed after 96 h. Levels of overexpressed Gβ_lγ₂ dimer were determined by immunoprecipitating HA-Gγ₂ and immunoblotting for Gβ_l and HA-Gγ₂. The effect of siRNA treatment on PhLP expression in HEK-293 cells was determined by immunoblotting whole-cell extracts for PhLP. Band intensities were quantified and expressed as a percentage of the lamin A/C sample. Bars and symbols in each panel represent the average±s.e. from three or four separate experiments (^*P<0.01). (B) The rate of nascent Gβ_lγ₂ dimer formation was determined using a radiolabel pulse–chase assay. Cells were pulsed for 10 min with [³⁵S]methionine followed by a chase with unlabeled methionine. Times indicate the sum of the pulse and chase periods. After the chase, Gβ_lγ₂ dimers were immunoprecipitated with an antibody to HA-Gγ₂. The dimers were resolved by Tris–Tricine–SDS–PAGE and radioactive protein bands were detected using a phosphorimager. Band intensities were quantified and molar ratios of Gβ_l to Gγ₂ were calculated. Lines represent a nonlinear least-squares fit of the data to a first-order rate equation. Values for t_1/2 are shown next to the graph.