Bca kit

LNCaP cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences and cultured in RPMI-1640 media (GE Healthcare Life Sciences HyClone laboratories, Logan, UT, USA) with 10% fetal bovine serum (FBS, Clark Bioscience, Richmond, VA, USA).
To detect the effect of triptolide on cell growth, LNCaP were plated on 96-well plate (1.5 × 10⁵/well) and treated with triptolide (100 nM). The viable cells were detected with SRB assay [8 (link)].
To detect the AR protein level, after being plated on 6-well plates, LNCaP cells were pre-treated with CHX (20 ng/mL) for 2 h and then triptolide (100 nM) was added to the media. After co-treatment with CHX and triptolide for 24 h, the cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, Haimen, Jiangsu, China) and the protein concentration was detected with BCA kit (Beyotime Biotechnology, Haimen, Jiangsu, China).
To detect the role of calpain in triptolide mediated AR degradation, calpain inhibitor ALLM and calcium chelator BAPTA-AM were used. After co-treatment with triptolide and ALLM (50 μM and 100 μM), PMSF (100 μM) or BAPTA-AM (10 μM) for 24 h, the cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, Haimen, Jiangsu, China) and the protein concentration was detected with BCA kit (Beyotime Biotechnology, Haimen, Jiangsu, China).

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.

Western blot analysis was performed as previously described [24 (link)]. Briefly, total protein was isolated from cells using the RIPA lysis buffer (Beyotime). After measuring the protein concentration using a BCA kit (Beyotime), a total of 40 μg protein was separated by 8% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto the polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany). Membranes were subsequently blocked with 5% skim milk at room temperature for 2 h, and incubated at 4°C overnight with primary antibodies: anti-CCT3 (Abcam, Cambridge, UK, ab225878, 1:2,000), anti-cyclin B1 (Abcam, ab181593, 1:2,000), anti-cyclin dependent kinase 1 (CDK1, Abcam, ab133327, 1:10,000), anti-Cleaved Caspase-3 (Abcam, ab2302, 1:500), anti-STAT3 (Abcam, ab119352, 1:5,000), anti-p-STAT3 (Abcam, ab76315, 1:5,000), anti-JAK2 (Cell Signaling Technology, Inc., MA, USA, #3230, 1:1,000), anti-p-JAK2 (Cell Signaling Technology, #4406, 1:1,000), and anti-GAPDH (Beyotime, AG019, 1:1,000). After incubation with corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime, A0208, A0216, 1:5,000) at room temperature for 2 h, membranes were visualized under a ChemiDoc XRS+ system (Bio-Rad). The protein bands were quantified using the Image J software (National Institutes of Health, Bethesda, USA).