Figure 3

G beta/gamma binding to cyclase(s): Western analysis and quantification A: Analysis of G beta bound to cyclase Western blot analysis of beta/gamma bound to cyclase(s) complex. Plasma membrane (50 μg protein) was incubated with isoproterenol or baclofen (200 nM) plus Mg (1 mM) and GTP (10 μM) for 10 min at 20°C. A 'pull down' experiment was performed in ice with Forskolin-agarose and solubilized membranes as described in 'materials and methods'. The material bound to the affinity beads was submitted to gel electrophoresis followed by Western blot analysis using anti-beta antibody. Membrane control (lane 1), isoproterenol (lane 2), isoproterenol + pertussis toxin (PTx) treatment (lane 3), baclofen (lane 4) and baclofen + pertussis toxin (PTx) treatment (lane 5). The arrow corresponds to MW = 35 kDa. B: Time course of association G beta /cyclase Same protocol as in A except that membranes were incubated with baclofen (left) and isoproterenol (right) at 200 nM. for 1.5 and 10 min before the cyclase complex was isolated by affinity chromatography. C: Quantification of G beta bound to cyclase (in A) To quantify the beta subunit bound to affinity beads, we used Bolton Hunter labeling of the affinity isolated material obtained under the conditions described in A, then carried out gel electrophoresis. The band about MW = 35 kDa was excised and counted. The bars represent 3 determinations +/-SE. * corresponds to values statistically different from control, P < 0.05. Lane 1: membrane control, lane 2: isoproterenol, lane 3: isoproterenol + pertussis toxin (PTx), lane 4: baclofen, lane 5: baclofen + pertussis toxin (PTx).