Bcl xl

Whole protein was extracted using RIPA lysis buffer (Beyotime, Beijing, China). The BCA Protein Quantification Kit (Tiangen, Beijing, China) was used to measure the protein concentration. The procedure was performed according to the method described in a previous study [14 (link)]. The primary antibodies used were: Oct4 (cat. no. 11263-1-AP, ProteinTech, Wuhan, China, 1: 1000), Nanog (cat. no. 67255-1-Ig, ProteinTech, 1: 3000), Sox2 (cat. no. WL03273, Wanleibio, Shenyang, China, 1: 1500), Cleaved caspase 3 (ab32042, Abcam, 1: 3000), Caspase 3 (cat. no. 66470-2-Ig, Proteintech, 1: 1000), Cleaved PARP (ab32561, Abcam, 1: 3000), PARP (cat. no. WL0326, Wanleibio, 1: 1500), p-STAT3 (Tyr705) (ab76315, Abcam,1: 1000), p-STAT3 (Ser727) (ab32134, Abcam, 1: 1000), STAT3 (cat. no. WL01836, Wanleibio, 1: 1500), c-Myc (cat. no. WL01781, Wanleibio, 1: 1500), Bcl-xl (cat. no. WL03353, Wanleibio, 1: 1500), and β-actin (cat. no. WL01372, Wanleibio, 1: 200).

After a 48-hour transfection period, the cells were collected, washed twice with PBS, added to radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China) for 30 min on ice and centrifuged at 12,000 rpm for 15 min at 4°C. The total protein extracted was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking in 5% skim milk for 2 h, the membrane was incubated with primary antibodies at 4°C overnight. Next, the membrane was incubated with horseradish peroxidase-conjugated goat anti-ribbit secondary antibodies at 1:2,000 for 2 h. The proteins were visualized by BeyoECL Star (Beyotime Biotechnology). The primary antibodies for ZEB1, E-cadherin and N-cadherin were purchased from Cell Signaling Technology (Massachusetts, MA, USA). The primary antibodies for Bcl-2, Bcl-xL, Bax, caspase-3, caspase-8 and caspase-9 were purchased from Wanleibio (Shenyang, China).