Ccd camera

Target tissues were homogenized in the RIPA lysis buffer. The supernatant was used for Western blot analysis. Protein concentrations were determined using the BCA assay regent. Thirty to sixty micrograms of protein lysates were separated on 6–12% sodium dodecyl sulfate‐polyacrylamide gels and then transferred to the PVDF membranes (Millipore). TBST (TBS and 0.1% Tween‐20) containing 5% nonfat milk or bovine serum albumin was used to block nonspecific binding for 2 h at room temperature. Then, the membranes were incubated with the primary antibodies against ERα, ERβ, E‐cadherin, and p53 (detailed information is shown in Table S1). The membranes were rinsed three times with TBST for 10 min and reincubated for 1 h at room temperature in blocking buffer with each HRP‐conjugated secondary antibody (1:5000), and then washed three times for 10 min each. Signals generated by enhanced chemiluminescence (Millipore) were recorded with a CCD camera (CLINX, Shanghai).

Cells were washed and lysed in RIPA buffer supplemented with protease/phosphatase inhibitor cocktail (5872, Cell Signaling Technology, CST). Protein lysates were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked in TBST (Tris-buffered saline containing 0.1% Tween-20) containing 5% nonfat dry milk or bovine serum albumin for 2 h before incubation with the desired primary antibodies at dilutions recommended by the manufacturers overnight. Then, the membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilutions). Signals generated by enhanced chemiluminescence (Millipore) were recorded with a charge-coupled device (CCD) camera (CLINX, Shanghai, China). Data are representative of at least three independent experiments. The following antibodies were used: LC3 (12741, CST), AMPK (5831, CST), p-AMPK (2535, CST), ULK1 (6439, CST), p-ULK1 (12753, CST), cleaved caspase-3 (9661, CST), caspase-3 (9662, CST), and β-actin (A5441, Sigma).

The total protein were extracted from target tissues and cells prepared with ice-cold lysis buffer (Biosharp). The supernatant was used for Western blot analysis. Protein concentrations were determined using a BCA protein kit (Beyotime Institute of Biotechnology, China). Samples containing equal amounts of protein were mixed with loading buffer containing 5% 2-mercaptoethanol and then heated for 10 min at 95°C. Twenty to forty micrograms of protein lysates were separated on 8–12 % sodium dodecyl sulfate-polyacrylamide gels and then transferred to the NC (Nitrocellulose) membranes (Millipore, Bedford, MA, USA). Tris Buffered Saline, with Tween-20 (TBST) containing with 5% nonfat milk or bovine serum albumin was used to block nonspecific binding for 2h at room temperature. Then, the membranes were incubated with the primary antibodies. Subsequently, the membranes were rinsed three times with Tris Buffered Saline (TBS) and 0.1% Tween-20 (TBS-T) for 10min and re-incubated for 1h at room temperature in blocking buffer with each Horseradish Peroxidase (HRP)-conjugated secondary antibody (1:5000), then washed three times for 10min each. Signals generated by enhanced chemiluminescence (Millipore) were recorded with a CCD camera (CLINX, Shanghai, China). Data are representative of at least three independent experiments.