Fig. 1

CBD disrupts the association of σ1R with the NR1 subunits of NMDA receptors. In vitro assay determining ligand activity for σ1R. NHS-activated Sepharose beads covalently coupled to a sequence of the NR1 subunit containing seven residues of the transmembrane region plus C0-C1-C2 cytosolic segments were incubated with excess σ1R (1:3). Unbound σ1R was washed out, and the NR1 C1-coupled σ1R was exposed to serial concentrations of the ligands under study. σ1R that remained attached to the NR1 subunits was then evaluated by SDS-PAGE and immunoblotting. a Inhibitory effects of the σ1R antagonists progesterone and BD106 and of CBD on the association of σ1R with the NR1 C1 subunit. The assays were performed in the presence of 50 mM Tris-HCl (pH 7.5), 0.2% CHAPS and 2.5 mM calcium. Representative blots are shown. The ED50 values were computed using the software SigmaPlot v.14. b The σ1R agonists PPCC, PRE084 and 4-IBP did not alter σ1R-NR1 associations but reduced the capacity of progesterone and CBD to disrupt such associations. PPCC reduced the capacity of CBD to inhibit the binding of σ1R to NR1 subunit in a concentration-dependent manner. The assays were performed twice, and each point was duplicated. *Significant difference with respect to the control group (DMSO or saline); φ significant difference with respect to the group receiving only CBD or progesterone; ANOVA, Dunnett’s multiple comparison, p < 0.05