Ab278053

Tissues and cells were harvested and lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich, St Louis, USA). After the proteins were extracted, the concentrations were measured using a BCA kit (Beyotime Biotechnology, Shanghai, China), and proteins were separated by 8%‒12% SDS-PAGE and blotted onto PVDF membranes (Millipore, Billerica, USA). After being blocked with non-fat milk for 1 h, membranes were incubated with specific primary antibodies targeting ELF4 (sc-515363; Santa Cruz, Santa Cruz, USA), FUT9 (60230-1-lg; Proteintech, Chicago, USA), CD44 (ab264539; Abcam, Cambridge, UK), and CD133 (ab278053; Abcam) overnight at 4°C. After wash with TBST 3 times, the membranes were incubated with the corresponding secondary antibodies for 2 h at room temperature. Finally, the ECL luminescent solution was evenly spread onto the PVDF membrane, and the signals were detected. Antibodies against β-actin (ab8227; Abcam), GAPDH (Cat#2118; Cell Signaling Technology, Beverly, USA), and tubulin (Cat#2146; Cell Signaling Technology) were used as the loading controls for western blot analysis.

The following antibodies were used for western blotting: rabbit monoclonal anti-CD24 (1:500, catalog number ab179821, Abcam, MA, USA), rabbit monoclonal anti-CD44 (1:500, catalog number ab189524, Abcam), rabbit recombinant multiclonal anti-CD133 (1:800, catalog number ab278053, Abcam), mouse monoclonal anti-Nanog (1:1000, catalog number ab173368, Abcam), rabbit monoclonal anti-OCT4 (1:500, catalog number ab200834, Abcam), mouse monoclonal anti-SOX2 (1:1000, catalog number ab79351, Abcam), rabbit polyclonal anti-DLG5 (1:1000, catalog number ab231283, Abcam), rabbit polyclonal anti-PRDM16 (1:1000, catalog number ab106410, Abcam), mouse monoclonal anti-ErbB-2 (1:500, catalog number sc-33684, Santa Cruz Biotechnology, CA, USA), and mouse monoclonal anti-β-actin (1:1000, catalog number A5441, Sigma-Aldrich, MO, USA). Other primary antibodies and secondary antibodies and experimental procedures for immunoblotting are provided in the supplementary experimental procedures^{10 (link)}.

The proteins of differently treated cervical cancer cells were extracted with RIPA Lysis Buffer (PP110; Protein Biotechnology Co., Ltd, China) according to the manufacturer's instruction. Then, the concentration of the proteins was determined using a BCA protein assay kit (PP202; Protein Biotechnology Co., Ltd, China). Subsequently, 30 µg proteins in each group were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gels were then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated in Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat milk for 1 h. Then, the PVDF membrane was incubated overnight with primary antibodies against NANOG (ab203919; Abcam, USA), OCT4 (ab200834; Abcam, USA), CD133 (ab278053; Abcam, USA), SOX2 (ab92494; Abcam, USA), and β-actin (ab8227; Abcam, USA). Then, the membrane was incubated with HRP-conjugated secondary antibody (ab205718; Abcam, USA) for 2 h at room temperature. The bands of individual proteins were visualized using a chemiluminescence (ECL) kit (PP404; Protein Biotechnology Co., Ltd., China).