Anti bmi1

The protein of glioma cells LN18 and U251 were isolated by RIPA buffer (P0013B; Beyotime) with protease inhibitors cocktail (C00001; TargetMol). Get an equal amount of protein sample from RIPA lysate for Western blot analysis. The protein was separated by SDS-PAGE, and then electro-transfer onto poly-vinylidene difluoride membranes (IPVH00010; Millipore). Then 5% skim milk was blocked for 1.5 hours, and then the primary antibodies were incubated overnight with anti-β-actin(1:10,000; #66009-1-Ig; Proteintech Group), anti-BMI1(1:2,000; #66161-1-Ig; Proteintech Group), anti-CD44(1:5,000; #60224-1-Ig; Proteintech Group), anti-Vimentin(1:5,000; #10366-1-AP; Proteintech Group), anti-ZEB1(1:1,000; 21544-1-AP; Proteintech Group), anti-bodies, respectively, at 4° C. Then incubate with the secondary antibody for 1 hour. Protein observation was done using ECL-Chemiluminescence kit (ECL-plus, Thermo Fisher Scientific, Inc.), and then detect the luminescence with a Protein Imager (Find-Do×6; Tanon). The relative gray level of western blot was measured by the ImageJ software for Microsoft Windows (National Institute of Health, Bethesda, MD, USA).

The experiment was carried out following our previously published protocol [13 (link)]. Anti-FOXD1 antibody was purchased from Genetex (1:1000; CA, USA). Anti-E-cadherin, anti-N-cadherin, and anti-vimentin antibodies were purchased from Cell Signaling Technology (1:1000; MA, USA); anti-CD44, anti-ALDH1A1, anti-BMI1, and anti-SNAI2 antibodies were purchased from Proteintech (1:1000; Wuhan, China). The gray value of the band was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).