Nr5a2 antibody

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)}

The FaDu (#HTB-43), SCC-4 (#CRL-1624), SCC-15 (#CRL-1623)and SCC-9 (#CRL-1629) cell lines were purchased from ATCC. FaDu, SCC-4, SCC-15 and SCC-9 cells were maintained in Dulbecco’s Modified Eagle Medium (#A4192101, Thermo Fisher) with 10% fetal bovine serum (#10099141, Thermo Fisher) in a 5% CO2 incubator at 37°C. GAPDH antibody (#ab8245, working dilution 1:3000) was purchased from Abcam; NR5A2 antibody (#22460-1-AP, working dilution 1:800) and p53 antibody (#60283-2-Ig, working dilution 1:1000) were obtained from Proteintech. siRNAs were purchased from RIBOBIO CO.,LTD. (GUANGZHOU, China). siRNAs were transfected into the cells by using the Lipofectamine 2000 (Invitrogen, USA).