Anti bax

Tissue samples and HCC cells were lysed using RIPA lysis buffer supplemented with PMSF (1:100, G-CLONE) to obtain total proteins. The proteins concentration was estimated using the BCA protein quantification kit (Solarbio). Equal amounts of protein were subjected to SDS-PAGE, proteins were then transferred onto PVDF membranes (PerkinElmer). After blocking with skimmed milk or BSA, the membranes were incubated with the primary antibodies overnight at 4 ℃. Primary antibodies were as follows: anti-EVA1A (1:500, Abcam), anti-TP53 (1:1000, OriGene), anti-BAX(1:2000, OriGene), anti-BCL-2 (1:2000, Bioss), anti-p-JAK2 (1:1000, Cell Signaling Technology), anti-p-STAT3 (1:1000, Cell Signaling Technology), anti-MMP-9 (1:1000, Cell Signaling Technology), anti-LC3 (1:1000, MBL), anti-p62 (1:1000, Proteintech), anti-Beclin1 (1:2000, Proteintech) and anti-β-actin (1:1000, Servicebio). And then the blot was incubated with peroxidase-conjugated sheep anti-rabbit IgG (1:3000, Servicebio). Protein bands were detected using the ECL system. Each independent experiment was performed at least three times.

Fetal bovine serum (FBS; US origin) was purchased from HyClone Laboratories Inc., South Logan, UT, USA. Anti-caspase-3, anti-caspase-8, anti-PARP, anti-BID, anti-Bax, anti-COX-2 and anti-beta-actin antibodies were purchased from OriGene Technologies, Inc., Rockville, MD, USA. Anti-p53, anti-Bcl-2 (NT), anti-caspase-9 and anti-iNOS antibodies were purchased from AnaSpec, Inc., Fremont, CA, USA. Anti-Cdc25C, anti-CDK1, anti-CDK2, anti-Cyclin A1 and anti-Cyclin B1 antibodies were purchased from Bioss, Inc., Woburn, MA, USA. Anti-p21 (WAF1, Cip1) was purchased from eBioscience, San Diego, CA, USA. Anti-cyclin D1 and anti-cdk-4 antibodies were purchased from Cell Signaling Technology, Inc., Danvers, MA, USA. Cell Proliferation Reagent WST-1 and the Annexin-V-FLUOS Kit were purchased from Roche Diagnostics, Mannheim, Germany. Alkaline phosphatase-conjugated anti-Rabbit secondary antibody was purchased from RockLand Immunochemicals Inc., Gilbertsville, PA, USA. Z-IETD-FMK (caspase-8 inhibitor) and Z-LEHD-FMK (caspase-9 inhibitor) were purchased from Abcam, Cambridge, MA, USA. All the other chemicals and solvents are of analytical or molecular biology grade and procured locally.

Total cell lysates were prepared from cell pellets using RIPA buffer (Solarbio, China) following the manufacturer’s protocol. After quantification of protein concentration, 40 µg of protein extracts was subjected to western blotting analysis. Different primary antibodies, including anti-p-ERK (1:1,000 dilution; ImmunoWay, USA), anti- ERK (1:1,000 dilution; ImmunoWay, USA), anti-cyclin D1 (1:5,000 dilution; Abcam, USA), anti-Bax (1:1,000; OriGene, China), and anti-β‑actin (1:1,000 dilution; Boster, China), were utilized in the analysis. Thereafter, the samples were incubated with various HRP-conjugated secondary antibodies (Boster Biological Technology, China). Immune complexes were visualized using the Enhanced Chemiluminescence (ECL) Kit (Thermo Fisher Scientific, USA), and band intensities were calculated using the Gel imaging system (Tanon, China).