Anti gapdh

The protein concentration was quantified by the BCA Protein Assay Kit (Beyotime) and normalized. Approximately 30 µg of protein extracts was analyzed by electrophoresis using a 12% SDS–PAGE gel and electroblotted onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% BSA and incubated with specific primary antibodies overnight at 4 °C with gentle rotation. A horseradish peroxidase-labeled secondary antibody was added, incubated for 1 h with shaking and visualized using an enhanced chemiluminescence reagent (Millipore). The primary antibodies included anti-GAPDH (#200306-TE4, 1:2 000), anti-FAK (#860324, 1:1 000), anti-p-FAK (#381143, 1:1 000), anti-ITGB1 (#R24729, 1:1 000), anti-ITGA5 (#R24725, 1:1 000), and anti-ITGB3 (#384730, 1:1 000) from Zen BioScience and anti-AKT (#4691, 1:1 000), anti-p-AKT (#4060, 1:1 000), anti-ERK (#4695, 1:1 000), and anti-p-ERK (#4370, 1:1 000) from Cell Signaling.

The extraction of membranous and cytoplasmic protein was performed according to the kit (KeyGen Biotech, Jiangsu, China) instructions. Membranes were incubated with the primary antibodies anti-MCT4 (G-9, IgG2bκ)/anti-MCT4 (G-7, IgG3κ) (Santa Cruz Biotechnology, USA, 1 : 1500), anti-PD-L1 (Proteintech Group, Chicago, USA, 1 : 1000), anti-GAPDH (Zen Bioscience, 1 : 1000), and rabbit polyclonal anti-Caveolin-1 antibody (1 : 200, cat. no. 340158, Zen-Bio, Chengdu, China) overnight at 4°C. Membranes were scanned by Tanon 5200 apparatus (Tanon, Shanghai, China) with ECL luminescence kit (Millipore, Massachusetts, USA).

DPCs were isolated by differential digestion as described above, after which total protein was extracted and normalized according to the manufacturer’s instructions. The primary antibodies were anti-APE1 (1:1,000; abcam, USA), anti-DMP1 (1:1,000; Santa cruz, USA), anti-DSP1-H (1:1,000; Santa cruz, USA), anti-OPN (1:1000; abcam, USA), anti-ALP (1:1,000; abcam, USA), anti-OSX (1:1,000; abcam, USA), anti-Axin (1:1000; abcam, USA), anti-Lef1 (1:1000; abcam, USA), anti-non-p (active) β-catenin (1:1000; CST, USA), anti-p-GSK-3β (1:1000; CST, USA), anti-P21 (1:1000; abcam, USA), anti-cyclin-D1 (1:1,000; Santa cruz, USA) and anti-GAPDH (1:10,000; Zen, China) used as internal control. Then, the membranes were rinsed with TBST (0.1% Tween-20 in 0.01 mol/L TBS), incubated with appropriate horseradish peroxidase conjugated secondary antibodies at 1:5000 (Santa Cruz, USA) at room temperature for additional 2 h, visualized by Image Quant LAS 4000 mini (GE, UK). Densitometry analysis on the bands was performed using the NIH image J software and normalizing the data to total protein levels (Supplementary Fig. 1. and Supplementary Fig. 2).

After treating with 50μM 7,8-benzoflavone for 48 hours, protein extraction was conducted utilizing RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors in HT-29 and HCT-116 cell lines. The Western blotting procedures adhered to established protocols as delineated in prior publications 30 (link). The primary antibodies, anti-CDK1, anti-IL1RN and anti-GAPDH, were procured from Zenbio, China.

Western blotting was performed following standard procedures. Anti-ACSL4 antibody (1:1,000, A16848, ABclonal, Woburn, MA, USA), anti-ferritin Heavy Chain (FTH) antibody (1:1,000; 381204, Zen Bio), anti-GPX4 antibody (1:5,000; ab125066, Abcam, Cambridge, UK), anti-ERK1/2 antibody (1:2,000; YT1625, Immunoway, Plano, TX, USA), anti-Phospho-Erk1/2 antibody (1:2,000; O1923, Cell Signaling, Danvers, MA, USA), anti-MEK1/2 antibody (1:2,000; D1A5, Cell Signaling, Danvers, MA, USA), anti-Phospho-MEK1/2 antibody (1:2,000; 41G9, Cell Signaling, Danvers, MA, USA) and secondary antibody (1:2,000; Beyotime, Jiangsu, China) were used to detect protein expression. Anti-GAPDH (Zen Bio) was used at a 1:2,000 ratio. Images were acquired by a Tanon 5500 imaging system (Tanon, Shanghai, China). The images were scanned with the Image J scanning software, and the data was expressed as relative values to sham or control values.

Cisplatin (HY-17,394) was purchased from MCE (New Jersey, USA). The TUNEL ApoGreen Detection Kit (40307ES20), the 1st Strand cDNA Synthesis Kit (11141ES60) and Hieff qPCR SYBR Green Master Mix (11201ES08) were purchased from YEASEN Biotechnology (Shanghai, China). The following primary antibodies were used in this study: anti-Bax (380,709), anti-collagen I (501,352), anti-α-SMA (380,653), anti-NOX4 (380,874), anti-TGF-β1(346,599), anti-p-smad2 (R26361), anti-p-smad3 (380,775), anti-smad2 (200,790), anti-smad3 (R25743). anti-beta tubulin (380,628), and anti-GAPDH (380,626), which were purchased from Zen BioScience (Chengdu, China). Rabbit anti-PCNA polyclonal antibody (bs-2007R) was obtained from Bioss (Beijing, China). Masson (G1340) and Sirus red stain (G1472) were obtained from Solarbio (Beijing, China). 8-OHdG (bs-1278R), 4HNE (bs-6313R), and 3NIT (bs-8551R) were purchased from Bioss (Beijing, China). Endogenous peroxidase blocker was obtained from Zhongshan Jinqiao (Beijing, China). Orthofluorescence microscope was purchased from ZEISS (Germany).

Cells were dissolved in RIPA buffer (10 mM Tris–HCl pH 7.5, 1% sodium deoxycholate, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, and 5 mM EDTA) plus a protease inhibitor). Protein concentrations were quantified using a BCA Protein Assay Kit (Beyotime Biotechnology). The same amount of protein (40 μg) was separated by SDS-polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylindene fluoride (PVDF) membranes (Immobilon-P^SQ). After transfer, the membranes were blocked with 5% skim milk powder in TBST buffer (50 mM Tris–HCl, 100 mM NaCl, and 0.1% Tween-20, pH7.4) at RT for 1 h and were incubated with the following antibodies at the indicated concentrations overnight at 4 °C. The antibodies included anti-GAPDH (Gene ID: 2597, ZEN-BIO); anti-STEAP3 (Cat No: 17186-1-AP, Proteintech); anti-SLC3A2 (Cat No: 66883-1-lg, Proteintech); anti-GPX4 (Cat No: 67763-1-lg, Proteintech); anti-Ferritin (Gene ID: 2512, Beyotime); anti-PDL2 (Gene ID: 58205, ThermoFisher). Proteins were visualized using ECL plus fluorescence detection reagent (Thermo Scientific, Waltham, MA).