Anti gapdh antibody

Protein was extracted from the proximal 1 cm portion of the colon tissue using RIPA lysis buffer containing protease inhibitor and quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Then, 20 μg protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocking for 1 h, the membranes were incubated with primary antibodies such as anti-Cytochrome C antibody (ab133504, abcam, Cambridge, UK); anti-Caspase 3 antibody (ab184787, abcam, Cambridge, UK); anti-Cleaved Caspase 3 antibody (ab214430, abcam, Cambridge, UK); anti-NLRP3 antibody (ab270449, abcam, Cambridge, UK); anti-Cleaved-IL-1ββ antibody (mAb#63124, CST, Boston, MA, USA); anti-phospho NF-kB p65 (Ser536) antibody (mAb#3033, CST, Boston, MA, USA); anti-phospho IκBα (Ser32) antibody (mAb#2859, CST, Boston, MA, USA); and anti-GAPDH antibody (AF0006, Beyotime, Shanghai, China) overnight at room temperature. Then, the membranes were incubated with the HRP-conjugated secondary antibody for 1 h. The color was developed with an electrochemiluminescence (ECL) reagent (Beyotime Biotechnology, Shanghai, China) and images were captured using the Bio-Rad gel imager (Bio-Rad, Hercules, CA, USA). The test method was based on previous papers from our research group [26 (link)].

For immunoprecipitation (IP), cells were suspended in lysis buffer (50 mmol/L Tris-Cl (pH 7.4), 150 mmol/L NaCl, 5% glycerol, 1% NP-40, 1 mmol/L EDTA), supplemented with 1 mM PMSF and 4 μg/mL protease inhibitor cocktail (Sigma). Lysates were centrifuged at 13,000 g for 10 min, and the supernatant was added to 2 μL indicated antibodies and 100 μL protein A agarose beads (Invitrogen) to incubate for 4 h at 4 °C. Afterward, protein A beads were washed with 250 mmol NaCl four times. For western blot, total proteins of liver tissue and LO2 cells were extracted with extraction buffer (RIPA). Nuclear proteins were extracted with the Nucleoprotein Extraction Kit protocol (Shanghai Sangon Biotech, China). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into a PVDF membrane (Millipore). The membranes were incubated with 5% BSA to block other contaminants, and then with primary antibodies. Immunoblotting was performed using anti-Bdh1 antibody (Abcam, UK), anti-Nrf2 antibody and anti-Histone H3 antibody (CST, USA), anti-cleaved caspase 3 antibody, anti-IL-18 antibody, anti-IL-1β antibody, anti-GAPDH antibody, and anti-β actin antibody (Beyotime, China).

Cells were lysed with RIPA lysis buffer (1% Triton X-100, 50 mM Tris-HCL, 135 mM NaCl, 0.1% sodium deoxycholate, 2 mM EDTA, 50 mM NaF, 2 mM sodium orthovanadate, 10 ug/ml of aprotinin, 10 ug/ml of leupeptin and 1 mM PMSF). Protein lysates were loaded onto SDS/PAGE gels and transferred to nitrocellulose membranes. Blots were blocked in 5% nonfat dry milk dissolved in PBS and incubated with specific antibodies overnight. Antibodies used in the present study were Anti-MT-CYB antibody (1:1000; abcam), anti-GAPDH antibody (1:1000, Beyotime). Protein expression was visualized with an ECL plus kit(Millipore Corporation, Billerica, MA, USA).

Cells were lysed using RIPA buffer (Beyotime Institute of Biotechnology), and the protein concentration was determined by using Pierce® BCA protein assay (Thermo Fisher Scientific, Inc.) using bovine serum albumin as a standard. Equal amounts of denatured proteins (30 μg) were subjected to SDS-PAGE electrophoresis and subsequently transferred to PVDF membrane (MiliporeSigma). After blocking the membranes with 5% defatted milk for 1 h, the membrane was incubated with the primary antibodies (see below) at 4°C overnight. After washing the membrane three times, the secondary antibody (see below) was added at room temperature for 1 h. Finally, the immunoblots were viewed using ECL Developer (Beyotime Institute of Biotechnology) and photographed with a gel imager (Bio-Rad Laboratories, Inc.). ImageJ software was used to calculate the relative gray values using β-actin as loading control. The antibody information was as follows: Primary antibody: anti-ALKBH5 antibody (1:1000, Cell Signaling Technology, Inc.), anti-MMP2 antibody (1:1000, Elabscience Inc.), anti-MMP9 antibody (1:1000, BOSTER Biological Technology Co. Ltd.), and anti-GAPDH antibody (1:5000, Beyotime Institute of Biotechnology); and secondary antibody: HRP conjugated anti-rabbit or -mouse secondary antibody (1:5000, MultiSciences Biotech Co., Ltd.).

Tetrandrine was acquired from Shanghai Ronghe Medical (Shanghai, China). Wright-Giemsa stain was obtained from Baso (Zhuhai, China). DCFH-DA was from Invitrogen (Carlsbad, CA). Acridine orange, 3-MA, DMSO, NAC, and Tiron were purchased from Sigma-Aldrich (St. Louis, MO). NBT was from Beyotime (Nantong, China). The FITC Annexin V Apoptosis Detection Kit I, CD14-FITC and CD11b-PE were obtained from BD Biosciences. Red blood cell lysis buffer, IL-3, IL6 and stem cell factor were kindly provided by Dr. Zan Huang (Wuhan University). MTS was acquired from Promega (Madison, USA). The antibody against LC3 was from Sigma-Aldrich (St. Louis, MO). The caspase-3, PARP, and ATG7 antibodies were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against CD14 and c-MYC were purchased from Proteintech Group Inc. (Chicago, IL). The anti-GAPDH antibody and the horseradish peroxidase (HRP)-conjugated secondary antibodies (goat–anti-rabbit and goat–anti-mouse) were acquired from Beyotime (Nantong, China).