Figure 4. Sodium vanadate treatment mimics PMT effects on Gα_q and Gα_i.

(A) Indo-1 AM labelled Swiss 3T3 cells were treated with 30 nM bombesin for 10 s (marked with solid bar beneath trace) and intracellular Ca²⁺ release was measured. Membrane proteins from Swiss 3T3 cells that were either (B) untreated or (C) treated with 30 nM bombesin for 1 min were separated by 2-D gel electrophoresis and Western blotted with anti-Gα_q/11 antibody. (D) Swiss 3T3 membrane proteins were incubated in the presence or absence of 30 nM bombesin for 20 min with or without 0.05 nM GTPγS. The proteins were then analysed for trypsin protection as described under Materials and Methods, and activated Gα_q/11 was separated by SDS PAGE and Western blotted with anti-Gα_q/11 antibody. Quantification of activated Gα_q/11 (lower panel) was determined by densitometric scanning and these data were analysed using factorial analysis of variance (ANOVA). The induction of activation shown is relative to the density of the band without GTPγS or bombesin. Bombesin significantly enhanced GTPγS binding to Gα_q/11 (* p = 0.002). Membrane proteins from Swiss 3T3 cells were incubated with (E) 1 mM sodium vanadate for 20 min at 37°C or (F) 1 mM sodium vanadate and 30 nM bombesin for 20 min at 37°C, proteins were separated by 2-D gel electrophoresis and Western blotted with anti-Gα_q/11 antibody. Membrane proteins from Swiss 3T3 cells were incubated (G) without or (H) with 1 mM sodium vanadate for 20 min at 37°C, proteins were separated by 2-D gel electrophoresis and Western blotted with anti-Gα_i-1-3 antibody. Samples from at least 3 independent membrane preparations were resolved with similar results.