3β diol

E15 mouse embryos were classified according to the sex or/and genotype. Cultured amygdala neurons were prepared and maintained as described^{7 (link)}. The medium was phenol red-free Neurobasal supplemented with B-27, N-2 and GlutaMAX I (Invitrogen). After 3 DIV the culture medium was replaced for 2 h by fresh medium devoid of supplements for Western blotting and quantitative real-time polymerase chain reaction analysis. Neuronal cultures were treated for 2 h with the following test compounds alone or in combination: E2 (10⁻¹⁰ M; Sigma-Aldrich), DHT (10⁻¹⁰ M; Steraloids), 3β-diol (10⁻¹⁰ M; Sigma-Aldrich), the selective ERα agonist propylpyrazoletriol (PPT, 10⁻⁷ M; Tocris Bioscience), the selective ERβ agonist dyarilpropionitrile (DPN, 10⁻⁹ M; Tocris), the GPER agonist G1 (10⁻⁷ M, Calbiochem), the selective ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 10⁻⁹ M; Tocris) and the selective AR antagonist flutamide (10⁻⁷ M; Tocris).

Four-Androstene-3, 17-dione (androstenedione), DHEA, DHT, 3α-DIOL, 3β-DIOL, flutamide (Flut), testosterone were obtained from Sigma Aldrich (St Louis, MO, USA). Steroids were dissolved in dimethyl sulfoxide (DMSO) at 50 mM and further diluted (the final concentration of DMSO in culture medium was 0.2 %). Lipopolysaccharide from Escherichia coli serotype 0127:B8 (LPS) was from Sigma Aldrich. All cell culture reagents and supplements were from Sigma. Antibodies against RACK1 and AR were from BD Biosciences (Franklin Lakes, NJ, USA), GRβ from Abcam (Cambridge, UK), and anti-human β-actin from Sigma Aldrich. AR siRNA and scrambled controls were from Cell Signaling Technology (Danvers, MA, USA). Electrophoresis reagents were from Bio-Rad (Hercules, CA, USA). All reagents were purchased at the highest purity available.