Aromatase

Total proteins were isolated from cells with lysis buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1% NP40; 2.5 mM sodium pyrophosphate; 0.02% sodium azide; 1 mM EGTA, 1 mM EDTA; 1 mM β-glycerophosphate; 1 mM Na₃VO₄; 1 mM PMSF; 1 μg/ml leupeptin). The lysates were centrifuged at 12 000r.p.m. for 30 min at 4 °C. The protein concentration was determined by Bradford dye method. Equal amounts (20–50 μg) of cell extract were subjected to electrophoresis in 8–12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) and transferred to PVDF or nitrocellulose membranes (Millipore, Darmstadt, Germany) for antibody blotting. The membranes were blocked and then incubated with CREB1 antibody (all from Cell Signaling Technologies, Boston, MA, USA), Aromatase, ERα, ERβ and NR5A2 antibody (all from Abcam, Cambridge, UK), β-actin antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) overnight at 4 °C. Subsequently, the membranes were incubated with an HRP-conjugated anti-mouse or -rabbit secondary antibody (Protein Tech Group, Chicago, IL, USA) at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence reagent (ECL) kit (GE Healthcare, Munich, Germany), according to the manufacturer's instructions. Blots were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).^{48 (link)}

Nuclear and cytoplasmic extracts were subjected to polyacrylamide gel electrophoresis using 4-12% Bolt gel system (Invitrogen), transferred to PVDF (Millipore) membranes, and immunoblotted. Antibodies used in Western blot are as follows: DVL-1 (D3570; Sigma), DVL-3 (SAB4200007; Sigma), Lamin (CS-4777; Cell Signaling), Aromatase (124776; Abcam), GAPDH (sc-47724; Santa Cruz Biotechnology, Inc) and β-actin (sc-47778; Santa Cruz Biotechnology, Inc). Membranes were incubated in 5% milk dissolved in TBST with primary antibody overnight at 4°C. Membranes were washed three time for 10 minutes each with TBST and probed with horseradish peroxidase-conjugated secondary antibodies in 5% milk/TBST for 1 hour at room temperature. Membranes were washed with TBST as previously described before visualization by enhanced chemiluminescence reagent (Thermo Scientific) on premium X-ray films (Phenix Research).

The primary antibodies were as follows: Olig2 (rabbit, AB9610, 1:500, Millipore; mouse, MABN50, 1:500, Millipore), MBP (rabbit, AB980, 1:750, Millipore), Adenomatus Polyposis Coli (APC/CC1) (mouse, OP80, 1:500, Calbiochem), GFAP (mouse, G3893, 1:1500, Sigma), Iba1 (rabbit, W1 W019-19741, 1:500, Wako), Arg-1 (goat, sc-18355, 1:100, Santa-Cruz), PDGFRα (rat, 558774, 1:500, BD Pharmingen), DHT (guinea-pig, GP-DHT1, 1 :200, Synabs), Neurofilament H (NF-H), Non-phosphorylated Smi-32 (mouse, 801701, 1 :300, Biolegend), Ki67 (mouse, 550609, 1:150, BD Pharmingen), Aromatase (rabbit, Ab18995, 1:200, Abcam), Claudin-5 (mouse, 35-2500, 1 :700, Invitrogen), pSTAT3 (Tyr705) (rabbit, 9145, 1: 500, Cell Signaling). AR is a home-made antibody (guinea-pig, Aa 283–298/Aa 406–420 from mus musculus AR Accession NP_038504.1; Eurogentec). The secondary antibodies were: goat anti-rabbit cyanine 3 conjugated (111165003, 1/250, Jackson Immunoresearch); goat anti-mouse Alexa 488 (A11029, 1:250, Thermo Fisher Scientific), goat anti-rabbit Alexa 633 (A21070, 1:750, Thermo Fisher Scientific), goat anti-rat Alexa 633 (A21094, 1:750, Thermo Fisher Scientific), goat anti-rabbit Alexa 488 (A32731, 1:350, Thermo Fisher Scientific); goat anti-guinea pig cyanine 3 conjugated (106165003, 1/500, Jackson Immunoresearch); donkey anti-goat Alexa 488 (A11055, 1:500, Thermo Fisher Scientific).