Anti ccng1

Protein lysates from HCC cells were subjected to Western blotting analysis by using anti-EZH2 (Abcam), anti-DNMT1 (Abcam), anti-DNMT3A (Abcam), anti-DNMT3B (Abcam), and anti-CCNG1 (Abcam) according to standard protocols as previously described [24 ,30 (link)]. Formaldehyde-fixed, paraffin-embedded sections were subjected to H&E staining and immunohistochemistry by using anti-Ki67 (Abcam) following the routine protocols.

Total proteins from tissues and cells were extracted by RIPA Lysis Buffer (Sangon Biotech Co., Ltd.). The obtained proteins were quantified using BCA Protein Assay kit (Sangon Biotech Co., Ltd.) and 30 µg proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels for 2 h. Next, the separated proteins were transferred onto the polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific, Inc.). These membranes were then immersed in 5% skimmed milk (Sangon Biotech Co., Ltd.) diluted with phosphate buffered saline and 0.1% Tween 20 (PBST) at room temperature for 3 h to prevent the non-specific protein binding, followed by incubation with primary antibodies overnight at 4°C. The primary antibodies were: anti-hexokinase 2 (anti-HK2; Abcam; cat. no. ab209847; 1:1,000), anti-CCNG1 (Abcam; cat. no. ab170389; 1:1,000) and anti-β-actin (Abcam; cat. no. ab8227; 1:3,000). Finally, the chromogenic reaction was performed by an HRP/DAB (ABC) detection kit (Abcam) after the incubation of anti-rabbit secondary antibody (Abcam; cat. no. ab205718; 1:5,000) for 1 h at room temperature. The gray level of each band was obtained by the statistical analysis of ImageJ software (National Institutes of Health) using β-actin as the endogenous reference.