Ab61241

Thirty milligrams of small intestinal tissue was homogenized and lysed in M-PER mammalian protein extraction reagent (Thermo Scientific, Rockford, IL, USA). Protein concentration was estimated using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). Cell lysates (50 μg) were loaded onto 5–10% gradient polyacrylamide gels. Proteins were electroblotted onto polyvinylidene difluoride membranes (Millipore, MA, USA) and immunolabeled using anti-SIRT1 antibody (1:800, ab28170, Abcam Biotechnology, Cambridge, MA, USA), anti-acetylated p53 antibody (1:600, ab61241, Abcam Biotechnology, Cambridge, MA, USA), anti-SOD2 antibody (1:1000, ab68155, Abcam Biotechnology, Cambridge, MA, USA), and antibodies against β-actin (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Enhanced chemiluminescence plus reagent (Boster Biotechnology Co., Wuhan, China) was used for chemiluminescent signal detection. Quantitative analysis was performed using Quantity One software version 4.6.2 (http://www.bio-rad.com/en-us/product/quantity-one-1-d-analysis-software).

Radio‐immunoprecipitation assay buffer (Beyotime, Shanghai, China) was applied to lyse the cells or tissues to obtain total protein. Then, the protein was quantified with the BCA Protein Assay Kit (Beyotime) following the conditions suggested by the manufacturer. A quantity of 40 μg of total protein was used for SDS‐PAGE followed by electrophoretic transfer onto polyvinylidene difluoride (PVDF) membranes. After blocking, the membranes were incubated with an anti‐GAPDH antibody (ab181603, 1:1000; Abcam, Cambridge, UK), rabbit polyclonal anti‐Fas antibody (ab82419, 1:1000; Abcam), rabbit polyclonal anti‐p53 antibody (ab61241, 1:1000; Abcam) or rabbit polyclonal Anti‐Egr1 antibody (ab208780, 1:1000; Abcam) in TBST containing 5% non‐fat milk overnight at 4°C. After washing three times with TBST, the membranes were incubated with a goat polyclonal to rabbit IgG H&L (HRP) pre‐adsorbed (ab7090; Abcam, Cambridge, MA) at a dilution of 1:2000 in phosphate‐buffered saline with 0.1% Tween 20 (PBST) for 1 hour at room temperature (RT). The membranes were subsequently washed three times with PBST, and the electrochemiluminescence plus system (Beyotime) was used to visualize the protein bands. The density of the immunoblot was analysed using the Lab Works 4.5 software (Ultra‐Violet Products, Cambridge, UK).