Anti oct4 11263 1 ap

Cellular protein extraction and immunoblot analysis were performed as previously described [71 (link),72 (link)]. Briefly, cellular proteins were extracted using a detergent containing CHAPS lysis buffer, separated by SDS-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane. The membrane was hybridized to primary antibodies: anti-ERCC1 (D61F5, Cell Signaling, Danvers, MA, USA), anti-OCT4 (11263-1-AP, Proteintech, Rosemont, IL, USA), anti-Nanog (EPR2027(2), Abcam, Cambridge, MA, USA), anti-SPC25 (ab20679, Abcam, Cambridge, MA, USA), and anti-GAPDH (AF7021, Affinity Biosciences, Cincinnati, OH, USA). The membrane was subsequently incubated with a secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotech, CA, USA). The membranes were developed using an ECL developing solution (Merck Millipore, MA, USA) followed by autoradiography. All immunoblot experiments were performed at least two times. The original blots are shown in the Figure S1, and one typical result is presented. The protein expression shown in each band was quantified after normalization to the GAPDH expression level. The error bars shown in the relevant figures indicated the standard deviation of the quantification results.

Cells were lysed on ice for 30 min in RIPA lysis buffer supplemented with protease inhibitors and a phosphatase inhibitor cocktail (Bimake), followed by centrifugation at 12,000 g for 10 min. The protein concentration was measured using a BCA Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein were resolved by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking, membranes were incubated with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. The immunoblots were visualized by ECL chemiluminescence (GE healthcare, Buckinghamshire, UK) using a Bio-Rad gel image analysis system. The following primary antibodies were used in this study: anti-ETV4 (sc-113, Santa Cruz), anti-HK2 (22029-1-AP, Proteintech), anti-LDHA (19987-1-AP, Proteintech), anti-PDK1 (3062 T, Cell Signaling Technology), anti-c-MYC (10828-1-AP, Proteintech), anti-OCT4 (11263-1-AP, Proteintech), anti-NANOG (14295-1-AP, Proteintech), anti-LIN28 (11724-1-AP, Ptoteintech), anti-SHH (ab53281, Abcam), anti-GLI1 (66905-1-Ig, Proteintech), anti-CXCR4 (60042-1-Ig, Proteintech), anti-β-actin (A1978, Sigma-Aldrich).