Western lumax light sirius hrp substrate reagent

Cells were digested with RIPA cell lysis buffer (Beyotime Biotechnology, Shanghai, China) with protease and phosphatase inhibitor cocktails (MCE, Shanghai, China) and were collected using a cell scraper. Total protein was extracted and quantified using the bicinchoninic acid method (Thermo Fisher Scientific). Proteins (20–30 μg) were resolved using 10% PAGE (Bio-Rad, Hercules, CA, USA), transferred to a nitrocellulose membrane (Pall Corporation, Port Washington, NY, USA) at 25 V for 30 min, and blocked for 1 h in 10% nonfat milk in 1× TBS/0.1% (v/v) Tween 20 at room temperature. Primary antibodies (GAPDH, 1:1,000, #5174; Cell Signaling Technology; β-actin, 1:1,000, #4970; Cell Signaling Technology; P1-HNF4A, 1:1,000, ab41898; Abcam; P2-HNF4A, 1:1,000, PP-H6939-00; R & D Systems; and CCL15, 1:1,000, ab219388; Abcam) were added and incubated overnight at 4 °C. Secondary antibodies (goat anti-rabbit IgG, HRP #7074 or goat anti-mouse IgG, HRP, 1:2,000, #7076; Cell Signaling Technology) were incubated at room temperature for 1 h. Signals were detected using Western Lumax Light Sirius HRP substrate reagent (ZETA-Life, San Francisco, CA, USA). All data were normalized to human β-actin or GAPDH. The bands were scanned using a ChemiDocXRS + Imaging System (Bio-Rad).

Tissues and cells were placed in ice-cold lysis buffer (50 mM Tris, 5 mM EDTA, 250 mM NaCl, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mM sodium orthovanadate), homogenized, and incubated in ice for 20 min. Samples were centrifuged at 11,000 g for 10 min, supernatants were collected, and the protein concentration was measured with a BCA Protein Assay Kit (Pierce). Equal amounts of protein from colonic tissues or cell lines were loaded onto SDS-PAGE gels. After electrophoresis and transference, membranes were blocked with 5% nonfat dry milk in TBST and incubated overnight at 4°C with primary antibodies (LC3B 1 : 400, Abcam; ATG16L1 1 : 100, Abgent; p65 1 : 200, Santa-Cruze; β-actin 1 : 2000, Abcam; GAPDH 1 : 2000, Abcam). Subsequently, membranes were incubated with secondary antibody (anti-rabbit or anti-mouse IgG, 1 : 2000, Abcam). Signals were detected using WesternLumaxLight Sirius HRP substrate reagent (ZETA-Life, San Francisco, CA, USA). The bands were scanned using a ChemiDocXRS+Imaging System (Bio-Rad, Hercules, CA, USA) and quantified by Image Lab v5.2 software (Bio-Rad).