Anti β tubulin

The total protein was extracted from the liver tissues using a Total Protein Extraction kit (Beyotime, Shanghai, China) and quantified using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein per sample (30 μg) were separated on a 10% SDS–PAGE and electroblotted onto nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA). After blocking with 5% non-fat milk for 2 h, the blots were incubated overnight with anti-VEGF (1:2000; Wanleibio, Shenyang, Liaoning, China), anti-Cyclin D1 (1:1000; Wanleibio, Shenyang, Liaoning, China), anti-NLRP3 (1:2000; Bioss, Beijing, China), anti-Wnt2 (1:1000; Abcam, Waltham, MA, USA), anti-β-catenin (1:10,000; Abcam, Waltham, MA, USA), anti-P-P65 (1:500; Santa, Dallas, TX, USA), anti-P65 (1:3000; Proteintech, Wuhan, Hubei, China), anti-caspase-1 (1:500; Santa, Dallas, TX, USA), anti-ASC (1:500; Santa, Dallas, TX, USA), anti-GSDMD (1:2000; Affinity, Liyang, Jiangsu, China), and anti-β-tubulin (1:1000; Wanleibio, Shenyang, Liaoning, China) primary antibodies at 4 ℃. The blots were washed with TBST and incubated with HRP-conjugated anti-IgG for 2 h. The positive bands were detected using an enhanced ECL reagent (Meilunbio, Dalian, Liaoning, China) on an AI600 System (GE Healthcare, Pollards Wood, UK). The relative protein expression was quantified using ImageJ software and normalized against the β-tubulin control.

SFN-Cys and SFN-NAC were purchased from Santa Cruz Biotechnology (USA). PTX (Taxol, as a brand name) was obtained from Selleckchem (USA). Anti-Caspase-3, anti-β-actin, anti-α-tubulin, anti-Tau and protein A/G PLUS agarose were purchased from Santa Cruz Biotechnology (USA). Anti-βIII-tubulin and anti-Caspase-7 were purchased from Abcam (USA). Anti-Hsp70, anti-XIAP, anti-ERK1/2 and anti-pERK1/2 (Thr202/Tyr204) were obtained from Cell Signaling Technology (USA). Anti-Stathmin1 was obtained from Sangon Biotech Co.Ltd. (Shanghai, China). Anti-cleaved-PARP and anti-β-tubulin were purchased from Wanleibio (Shenyang, China). Annexin V-FITC/PI apoptosis assay kit was purchased from NeoBioscience (Shenzhen, China). Recombinant human Caspase-3 was purchased from Sino Biological Inc. (Beijing, China).