Mouse anti flag

The cells treated with transfection and 4 Gy IR were harvested and lysed in a lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 100 mmol/L NaCl, 50 mmol/L sodium fluoride, 1% Nonidet P-40, 1 mmol/L dithiothreitol, 1 mmol/L Na₃VO₄, 1 mmol/L Microcystin-LR, 1 mmol/L phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, and 10 mg/mL aprotinin). The lysates were incubated with antibodies/protein G-conjugated agarose beads (Millipore, USA). The antibodies used were mouse anti-ERβ (Abcam, Cat NO.: ab288) and mouse anti-Flag (ABclonal, Cat NO.: AE004). The precipitates were washed six times with ice-cold lysis buffer, resuspended in PBS, followed by western blot analysis. For western blot analysis, the following antibodies were used: primary antibody is rabbit anti-ERβ (Proteintech, Cat NO.: 14007–1-AP) and rabbit anti-CLPTM1L (ABclonal, Cat NO.: A10468); secondary antibody is IPKine HRP mouse anti-rabbit IgG light chain (Abbkine, Cat NO.: A25022). The GST pull-down was performed according to published protocols [58 (link)]. Glutathione beads were recovered by a brief centrifugation and washed six times with lysis buffer, followed by western blot analysis.

For western blot analysis, total protein lysate was extracted from tissues or cells with RIPA buffer (Solarbio) after 2 days of IR. For relative experiments, the nuclear or cytoplasmic protein was extracted from cells using Nuclear Protein Extraction Kit (Solarbio). The protein samples were subjected to SDS-PAGE and then transferred to a nitrocellulose membrane, blocked with 5% non-fat milk, and incubated with primary antibodies for 2 h at room temperature. Primary antibodies used were mouse anti-GAPDH (Proteintech, Cat NO.: HRP-60004), rabbit anti-CLPTM1L (ABclonal, Cat NO.: A10468), mouse anti-ERβ (Abcam, Cat NO.: ab288), rabbit anti-CDC25A (Proteintech, Cat NO.: 55031–1-AP), rabbit anti-c-Jun (Cell Signaling, Cat NO.: 60A8), mouse anti-BCL2 (Cell Signaling, Cat NO.: 15071), rabbit anti-Histone H3 (Proteintech, Cat NO.: 17168–1-AP) and mouse anti-Flag (ABclonal, Cat NO.: AE004). Then, treated with secondary antibody diluted in PBS at room temperature for 1 h. Membranes were washed in PBS-T and bound antibody was detected by enhanced chemiluminescence system Western Blotting Detection Reagents (Amersham Biosciences, Buckinghamshire, UK). All experiments were repeated 3 times.

The cells were lysed in 1× RIPA lysis buffer to acquire the total cellular proteins. The supernatants were collected and then subjected to western blotting analysis. In brief, the proteins were first separated by SDS-PAGE and then transferred to 0.2 mm PVDF membranes, which were blocked for 1 h. The membranes were then incubated with specific primary antibodies overnight and subsequently with secondary antibodies for 1 h. The protein bands were detected using the Enhanced chemiluminescent (ECL) detection reagent (Bio-Rad, USA). The antibodies used in this study were as follows: rabbit anti-KDM4C (Bethyl Laboratories, A300-885A, 1:1000, USA); rabbit anti-GAPDH (Servicebio, GB11002, 1:1000, China); mouse anti-Flag (ABclonal, AE005, 1:1000, China); and rabbit anti-ZEB1, anti-Snail, anti-β-Catenin, anti-Vimentin (Cell Signaling Technology, #9782, USA).