Anti cse

Proteins extracted from ileal tissues and cells were harvested on ice and homogenized in RIPA lysis buffer containing 1% PMSF, 1% protease inhibitor cocktail, and phosphatase inhibitor cocktail. The protein (30 μg) was separated by SDS-PAGE and transferred onto NC membranes using the Trans-Blot Turbo system. The filters were blocked with 5% skimmed milk in Tris-buffered saline and Tween-20 (TBST) for 1 h and then incubated overnight at 4°C with primary antibodies, which include anti-FXR (1:2,000; R&D Systems; cat: A9033A), anticystathionine-γ-lyase (anti-CSE; 1:2,000; Abnova; cat: H00001491-M03), anti-NF-κB (1:1,000; Cell Signaling Technology; cat: 4764), and anti-β-actin (1:2,000; Santa Cruz Biotechnology; cat: 1313). After being washed with TBST, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) for 1 h at room temperature, and ECL reagent kits (Millipore, Billerica, Mass) were used for protein detection. The signals were quantified using ImageJ software (NIH, Bethesda, Md) and the values were normalized to β-actin and presented as the mean ± standard error of the mean (SEM).