Ab1500771

Total protein was extracted using radioimmunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China). Equal protein for each sample was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electrophoretically transferred to polyvinylidene fluoride membranes (GE Healthcare, Piscataway, NJ, USA). Subsequently, membranes were blocked with 5% skim milk solution at room temperature for 1 h. Protein expression was detected through incubation of the membranes with indicated primary antibody at 4°C overnight. After being washed three times for 10 min each, membranes were incubated with Goat polyclonal Secondary Antibody to Rabbit IgG-H&L (ab1500771; 1:2,000 dilution; Abcam, Cambridge, MA, USA) for 1 h at room temperature. ECL Western Blotting Detection Kit (Solarbio) and Image J software (National Institutes of Health, Bethesda, MD, USA) were used to visualize and quantify protein expression, correspondingly. The primary antibodies used were proliferating cell nuclear antigen (PCNA; ab92552; 1:1,000 dilution; Abcam), Cleaved-caspase-3 (Cleaved-cas 3; ab2302; 1:1,000 dilution; Abcam), matrix metalloproteinase 2 (MMP-2; ab97779; 1:1,000 dilution; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ab181602; 1:3,000 dilution; Abcam).

Briefly, HCC cells or tissues were lysed in ice-cold Radio-Immunoprecipitation buffer (Sigma-Aldrich) containing 1% Triton X-100, 62.5 mM Tris-HCl (pH 6.8), and the cocktail of proteinase/phosphatase inhibitors. After the protein concentration was quantified, 50 μg of protein was seperated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Next, the wet electrophoretic transfer method was performed to transfer protein onto the nitrocellulose filter membranes (Millipore, Billerica, MA, USA). Subsequently, the membranes were probed with antibodies at 4℃, such as anti-glucose transporter type 1 (GLUT1; ab181602; 1:1,000 dilution; Abcam, Cambridge, MA, USA), anti-RNF38 (1:1,000 dilution, Boster, Wuhan, China), and anti-GAPDH (1:2,000 dilution, Boster, Wuhan, China). After overnight, the membrane was probed with goat anti-rabbit polyclonal secondary antibody (ab1500771; 3,000 dilution; Abcam) for 2 h. Then, protein signals were visualized under the Bio-Rad ChemiDoc MP imaging system (Bio-Rad).