Ecl chemiluminescent substrate reagent kit

Western blot experiments were performed as previously reported (Xue et al., 2017 (link); Qin et al., 2018 (link)). In brief, 3 × 10⁵ cells were cultured in 6-cm dishes treated with specific concentrations of SDL-1 (0, 10, 20, and 40 μM) for 24 h. And then all cells were lysed in RIPA lysis buffer (Absin Bioscience Inc., Shanghai, China) containing protease inhibitor mixture (PMSF). The cell lysates were quantified by the BCA Protein Assay Kit (Absin, Shanghai, China). Equal amounts of protein were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, United States). Block the membranes in 5% skim milk at room temperature and incubated with primary antibody overnight at 4 °C followed by incubation with appropriate secondary antibody at room temperature. Antibodies used are as follows: STAT3 antibody (Cell Signaling Technology, #12640S), p-STAT3 antibody (Cell Signaling Technology, #49081S), and GADPH antibody (Proteintech, #60004-1-Ig). The protein bands were detected by ECL Chemiluminescent Substrate Reagent Kit (Biosharp, Hefei, China) and scanned using the ImageQuant 800 (Amersham, United Kingdom).

The methodology used here was described elsewhere [25 (link)]. In brief, the total protein was extracted from cells using RIPA buffer containing phosphatase and protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). The cell lysates were evaluated for their protein concentrations, resolved by SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Germany). The membranes were blocked in 5% skim milk and incubated with primary antibody followed by incubation with appropriate secondary antibody. ECL Chemiluminescent Substrate Reagent Kit (Biosharp, Hefei, China) was used to detect protein bands.