Sox2 antibody

The experimental group was defined using a predetermined experimental drug concentration (single or combination), while the drug-free medium was defined as a control group, with each group acting on the cells for 48 hours. After treatment, cells were collected for lysis. Protein concentration was determined using the bicinchoninic acid (BCA) Protein Assay Kit (Keygen, Nanjing, China). Samples of 20 µg of protein were collected, resolved in a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. Then, 5% blocking buffer [5% skimmed milk powder + tris-buffered saline with Tween 20 (TBST)] was applied to the membrane for 60 minutes at room temperature. Subsequently, the membrane was incubated with primary antibodies, including Sox2 antibody (Proteintech, 11064-1-AP), Oct4 antibody (Abcam, Cambridge, UK; ab200834), beta-actin (Proteintech, 66009-1-Ig), Bcl2 antibody (Abcam, mab182858), Bax antibody (Proteintech, 50599-2-Ig), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Proteintech, 10494-1-AP), Nanog antibody [Cell Signaling Technology (CST), Danvers, MA, USA; 4903T] overnight at 4 ℃. The next day, secondary antibodies were added and the membranes were washed and visualized using ChemiDoc TMXRS+ (Bio-Rad, Hercules, CA, USA) after 60 minutes of incubation at room temperature.

IGF2BP1 antibody (Proteintech, Rosemont, IL, USA), SOX2 antibody (Proteintech), GAPDH antibody (Proteintech) and α-tubulin antibody (Proteintech) were used in our western blotting analyses. We determined the protein concentration with the bicinchoninic acid protein concentration determination kit (Beyotime Biotech Inc.), and then PBS and 5 × loading buffer were added for quantification of the protein concentration to 2 ug/uL. After denaturation at 100°C for 15 minutes, separate the protein with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred protein to a polyvinylidene difluoride membrane. Next, seal the membrane with 5% skimmed milk, and primary antibodies were added, and then incubation of the membrane overnight at 4°C. The membrane was washed with Tris buffered saline with Tween 20 (TBST) for 10 minutes 3 times, and added the secondary antibody (1:8000, Proteintech) and incubated the membrane for 2 hours at room temperature, on the next day. And then wash the membrane with TBST for 10 minutes 3 times. Analyze the protein bands through the hypersensitive ECL chemiluminescence kit (NcmECL UItra; Sliding Biotech, Shanghai, China). ImageJ software was utilized to protein quantification.