Antibodies against α tubulin

To prepare tissue lysates of the inflamed distal colon, tissues (1.0 g) were disrupted and homogenized in 3.0 mL of ice-cold radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 150 mM NaCl, 0.3 μM aprotinin, 1 μM pepstatin, and 1 mM phenylmethylsulfonyl fluoride]. After incubation on ice for 30 min, the homogenates were centrifuged at 10,000× g at 4 °C for 10 min. Protein concentrations in the supernatants obtained by the above lysis process were determined using a BCA Protein Assay kit according to the manufacturer’s instructions (Pierce Biotechnology, Rockford, IL, USA). Tissue lysates were electrophoretically separated on 10% SDS-PAGE gels. Cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) proteins were detected using the following antibodies: Anti-COX-2 antibody (4842S, Cell Signaling Technology, Danvers, MA, USA), anti-iNOS (NOS-2) antibody (sc-7271, Santa Cruz Biotechnology, Dallas, TX, USA). Signals were visualized using the Supersignal chemiluminescence substrate (Pierce, Rockford, IL, USA). Experiments were normalized using antibodies against α-tubulin (Santa Cruz Biotechnology, city, Dallas, TX, USA).
Western blot images were quantified using Image Lab software (version 5.2 build 14, Bio-Rad, Hercules, CA, USA). The quantification results are expressed as the mean of quantified values (n = 5).

Proteins were extracted from cell lysates and quantified by the Bradford assay, then separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (EMD Millipore). Membranes were incubated with primary antibodies at 4°C overnight. The next day, after incubation with peroxidase‐coupled anti‐mouse or anti‐rabbit IgG at 37°C for 2 hours, protein levels were visualized by electrochemiluminescence. Antibodies against SIRT4 (1:40 000), STAT3 (1:2000), MYC (1:1000), and histone H3 (1:6000) were purchased from ProteinTech. Antibodies against p‐STAT3 Y705 (1:2000), CCND1 (1:1000), and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; 1:1000) were purchased from Cell Signaling Technology. Antibodies against α‐tubulin were purchased from Santa Cruz Biotechnology (1:1000). S3I‐201 was purchased from Selleck. Interleukin (IL)‐6 was purchased from Sino Biological.