Fig. 3: Antibody-capture [35S]GTPγS binding scintillation proximity assay (SPA).
A Schematic representation of the SPA methodology, created using BioRender. SPA allows the determination of the activation level of different Gα subunit subtypes present in postmortem brain tissue thanks to the selective immunoprecipitation of each of them and their coupling to protein A-coated polyvinyltoluene SPA beads. Modulation of specific [35S]GTPγS binding to Gαi1, Gαi2, Gαi3 and Gαq/11 proteins by 10 μM Nitro-I (B), Met-I (C), Otava 3575001 (OTV1) (D) and Otava 3736689 (OTV2) (E) in human prefrontal cortex both in the absence (colored bars) and in the presence (white bars) of the 5-HT2AR antagonist MDL-11,939. Basal values of specific [35S]GTPγS binding to the different G proteins are expressed as 100% and stimulatory/inhibitory effects on the respective basal are shown. Individual dots represent different assays 4–6 carried out for each subunit/condition performed in duplicate or triplicate. White dots represent data for the drug-alone condition, and black dots represent data for the drug + MDL condition (B–E). #p < 0.05 vs 100%; $p < 0.05 vs incubation in the presence of 1 µM MDL-11,939 (t-test). Modulation of specific [35S]GTPγS binding to Gαi1-, Gαi2-, Gαi3- and Gαq/11 proteins by 10 μM Nitro-I (F), Met-I (G), OTV1 (H) and OTV2 (I) in brain cortex tissue from WT (colored bars) and 5-HT2AR KO mice (white bars). #p < 0.05 vs 100%; $p < 0.05 WT vs 5-HT2AR KO (t-test). Basal values of specific [35S]GTPγS binding to the different Gα proteins are expressed as 100%, and stimulatory/inhibitory effects are expressed as % of the respective basal activity. Individual dots represent the different assays (3–5) for each subunit performed in duplicate or triplicate. White dots represent data for WT mice and black dots represent data for KO mice (F–I).
