Pvdf or nitrocellulose membranes

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)}

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 b-glycerophosphate; 1 mM Na₃VO₄; 1 mM PMSF; 1 μg/mL leupeptin). The lysates were centrifuged at 12,000 rpm for 30 min at 4°C. The protein concentration was determined by Bradford dye method. Equal amounts (20 to 50 μg) of cell extract were subjected to electrophoresis in 6-15% 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 Flag, HSPA5, JNK1/2, p-JNK1/2 and β-actin antibodies (all from Cell Signaling Technologies, Massachusetts, USA), MMP2 and MMP9 (all from Abcam), FOXM1 (C-20) (Santa Cruz Biotech) overnight at 4°C. Subsequently, the membranes were incubated with a HRP-conjugated anti-mouse or -rabbit secondary antibody (Protein Tech Group, Chicago, IL) 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 [47 (link)]. Bands were quantified using Image J software (Fujifilm, Tokyo, Japan) and normalized to a loading control.

Total proteins were isolated from cells with RIPA lysis buffer. The protein concentration was determined by Bradford dye method. Equal amounts (20 to 50 μg) of cell extract were subjected to electrophoresis in 6–15% 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 Cathepsin B, Cathepsin D, COX IV, Cytochrome c, phospho-EGFR (Tyr1068), EGFR, phospho-AKT (Ser473) and Actin antibodies (all from Cell Signaling Technologies, Massachusetts, USA), γ-tubulin and Ras (all from Abcam), AKT (Santa Cruz) overnight at 4°C. Subsequently, the membranes were incubated with a HRP-conjugated anti-mouse or -rabbit secondary antibody (Protein Tech Group, Chicago, IL) at RT 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.