Westernbright ecl reagent

Western blotting was conducted as described previously [39 (link)]. Lysed samples were measured using bicinchoninic acid (Thermo Scientific, Waltham, MA, USA) and bovine serum albumin was used as the standard for protein concentrations. Samples were prepared in a gel buffer [12.5 mM Tris buffer (pH 6.8), 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2-mercaptoethanol, and 0.2% bromophenol blue] and kept at 100 °C for 5 min. SDS-polyacrylamide gel electrophoresis containing with 8% to 14% acrylamide was used to separate equal concentrations of protein. Using a wet transfer system, the gels were transferred to polyvinylidene difluoride membranes at 90 V for 90 min. Membranes were immediately incubated in blocking buffer [10 mM Tris buffer (pH 7.5), 100 mM NaCl, 0.1% Tween 20, and 5% non-fat milk]. Blotting was done at 25 °C for 30 min and then membranes were incubated with specific primary antibody at 4 °C for 16 h. The secondary antibody was then added followed by an HRP conjugated anti-rabbit, anti-goat antibody, or anti-mouse antibody at 25 °C for 1 h. Antibody labeling was used to detect antibodies with WesternBright^TM ECL reagent (Advansta, Menlo Park, CA, USA).

Protein extraction and Western blot analysis were conducted as previously depicted [93 (link)]. Cells were rinsed in precooled PBS and proteins were extracted using RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8, 0.5% sodium deoxycholate, 1% NP-40, 0.2% SDS) containing protease inhibitor cocktail (EDTA free) (MedChemExpress, HY-K0011). After centrifugation (13,000× g, 10 min), total proteins were quantified with BCA Protein Assay Kit (CWBIO, Beijing, China, CW0014). Equal amounts of protein samples (30−80 μg) were separated on SDS-PAGE gels and then transferred to 0.45 μm PVDF membranes (GE Healthcare, Erlangen, Germany, A29280264). After incubation of the membranes with primary antibodies (1:1000) at 4 °C overnight, the samples were incubated with the secondary antibodies (1:5000) conjugated with horseradish peroxidase for 1 h at room temperature. The blots were visualized with WesternBright^TM ECL reagent (advansta, Bering Dr, San Jose, CA, USA, 191026-11) using ChemiDoc^TM XRS+ (Bio Rad, Berkeley, CA, USA).

Proteins from BC cells were lysed with RIPA buffer containing Complete Easy pack protease inhibitor cocktail tablets (04693116001, Roche, Mannheim, Germany). Protein concentration was determined using BCA Assay (#23227, Thermo Scientific, Rockford, IL, USA [47 (link)]) following the manufacturer’s instructions. Antibodies used in western blot were used to target E2F1 (A300-765A, Bethyl Laboratories, Montgomery, TX, USA), RAD54L (ab10705, Abcam), and β-Actin (#4967, Cell Signaling, Danver, MA, USA). Immunoreactivity was detected using a WesternBright^TM ECL reagent (K-12045-D50, Advansta, CA, USA) with HRP conjugates. Films were exposed at multiple time points to ensure that images were not saturated (47410 19291, Fuji Medical X-Ray Film, Tokyo, Japan). A ChIP assay was performed according to previous studies [30 (link)]. The indicated ChIP-qRT PCR primer sets (Table S1) were used to amplify DNA fragments by qRT-PCR.