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Figure 5 | BMC Cell Biology

Figure 5

From: Simultaneous stimulation of GABA and beta adrenergic receptors stabilizes isotypes of activated adenylyl cyclase heterocomplex

Figure 5

Covalent crosslinking of cyclase complex and quantification of G beta in the 'pull down' material. A: cross linking of cyclase complex Membranes were incubated with baclofen (200 nM) + isoproterenol (200 nM) in the presence of GTP (10 μM) + EDTA (1 mM) with or without GTPγS (10 μM) plus Mg (5 mM). Membranes were then placed in ice with an excess of GDP (1 mM) and Mg (5 mM) (t = 0), then were solubilized at different times (1, 5 and 15 min). A 'pull down' of the cyclase(s) complex was carried out with Forskolin-agarose beads and the material bound to the affinity beads was cross-linked with linkers (see 'materials and methods'). The molecular complexes were separated by gel electrophoresis and analyzed by Western blot using anti-beta antibody. left: bac + iso at 1, 5 min. Right: bac + iso + GTPγS at 1, 5 and 15 min respectively. Arrows correspond to MW > 200, 80 and 35 kDa respectively. B: time course dissociation Gbeta /cyclase: quantification of Gbeta at 1 and 10 min Quantification of beta/gamma in cyclase(s) complex, 1 min and/or 10 min after a large excess of GDP was added. Membranes were incubated with isoproterenol (200 nM) and/or baclofen (200 nM) for 20 min at 20°C, according to the same protocol as in figure 4 then placed in ice. Large excesses of GDP (1 mM) and Mg (5 mM) were added. The membranes were solubilized after 1 min and/or 10 min and the cyclase(s) complex was isolated by Forskolin-agarose. To quantify the beta subunit bound to the affinity beads, we carried out a Bolton Hunter labeling of the affinity isolated material followed by gel electrophoresis. Instead of Western blot analysis, gels were dried and the band about MW = 35 kDa was excised and counted. The bars represent 3 determinations +/-SE. * and ** correspond to P < 0.1 and P < 0.05 respectively compared to iso and/or bac. Lane 1:isoproterenol, lane 2: baclofen, lane 3: isoproterenol + baclofen, lane 4: isoproterenol + baclofen + GTPyS, lane 5: isoproterenol + baclofen + CTx, lane 6: CTx, lane 7: GTPyS

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