Anti β actin mouse monoclonal antibody

Antibodies used in the present study were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.): rabbit anti-Glut1 monoclonal antibody: (#12939); rabbit anti-Hexokinase 2 (HK2) monoclonal antibody: (#2867); rabbit anti-LDHA monoclonal antibody: (#3582); total PARP and cleaved PARP: (#9532) mouse anti-β-actin monoclonal antibody: (#3700). Imatinib mesylate, DAPI, 2-deoxyglucose (2-DG), and Oxamate were purchased from Sigma–Aldrich (Shanghai, China).

All antibodies were used for Western blotting. Rabbit anti-MLKL monoclonal antibody (1:1000; Abcam) was used to verify clones with MLKL deficiency while rabbit anti-GSDME-N-terminal monoclonal antibody (1:1000; Abcam) was used for detecting cleavage of GSDME. Mouse anti-β-actin monoclonal antibody (1:1000; Cell Signaling Technology) was used to indicate the loaded protein amount.

RIPA cell lysate in suitable volume was added to the cells. After centrifugation, by the bicinchoninic acid (BCA) protein concentration assay kit (Beyotime, Shanghai, China), we got the concentration of the protein in the supernate. Before being diluted by a 5× Loading Buffer, the samples were boiled with water bath lasting for 5 minutes. Then, we used SDS-PAGE to separate the protein and transferred them to a PVDF membrane (Millipore, USA). Since being incubated with primary antibody, including mouse anti-β-actin monoclonal antibody (diluted 1 : 1000; Cell Signaling Technology), mouse anti-claudin-4 monoclonal antibody (diluted 1 : 500; Invitrogen), rabbit anti-ZO-1 polyclonal antibody (diluted 1 : 250; Invitrogen), rabbit anti-ZONAB polyclonal antibody (diluted 1 : 1000; Invitrogen), and goat secondary antibody conjugated to horseradish peroxidase (diluted 1 : 5000; Sangon, Shanghai, China), ECL luminescence solution (Sigma, St. Louis, MO) was added as luminous substrate, and the band intensity was analyzed by a digital scanning imaging system.

T24 and 5637 cells and tumor tissues were lysed using RIPA protein extraction reagent (Beyotime Institute of Biotechnology) supplemented with 1% protease inhibitor cocktails (Roche Applied Science). Protein concentration was measured using the BCA assay (Beyotime Institute of Biotechnology; cat. no. P0012). Equal amounts of proteins (20 µg/lane) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). Following blocking with 5% non-fat milk at 4°C overnight, the membranes were probed with rabbit anti-HDAC2 monoclonal antibodies (1:2,000; Cell Signaling Technology, Inc.; cat. no. 57156s), mouse anti-β-actin monoclonal antibodies (1:3,000; Cell Signaling Technology, Inc.; cat. no. 3700), or rabbit anti-GAPDH (1:5,000; cat. no. sc-25778; Santa Cruz Biotechnology, Inc.) antibodies at room temperature for 1 h, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:4,000; OriGene Technologies, Inc.; cat. no. ZDR-5307, goat anti-mouse IgG/HRP and cat. no. ZDR-5306, goat anti-rabbit IgG/HRP) at room temperature for 1 h. Enhanced chemiluminescence reagent (Merck KGaA) was used to detect the signal on the membrane (Beijing Transgen Biotech Co., Ltd.).

The protein levels of CBS and CSE in the kidney were measured by Western immunoblotting analysis. In brief, kidney proteins (20 μg) were separated by electrophoresis in 10% SDS polyacrylamide gels. Proteins in the gel were transferred to a nitrocellulose membrane. The membrane was probed with (Abe and Kimura 1996 (link)) mouse anti‐CBS monoclonal (1:3,000; Abnova Corporation, Taipei, Taiwan) or rabbit anti‐CSE monoclonal antibodies (1:3,000; GeneTex, Irvine, CA) for rat proteins. HRP‐conjugated anti‐mouse or anti‐rabbit IgG antibodies (Cell Signaling Technology, Danvers, MA) were used as the secondary antibodies (1:5,000). The corresponding protein bands were visualized using enhanced chemiluminescence reagents and analyzed with a gel documentation system (Bio‐Rad Gel Doc1000). To confirm the equal loading of proteins for each sample, the same membranes were reprobed with mouse anti‐β‐actin monoclonal antibodies (1:5,000, Cell Signaling Technology). Proinflammatory factors (MCP‐1, IL‐6) in the kidney and plasma as well as plasma neutrophil gelatinase‐associated lipocalin (NGAL) were measured using the MesoScale Discovery electrochemiluminescence platform (Rockville, MD).