Tumor necrosis factor (tnf)

The extraction of total proteins was performed with cells at a concentration of about 2.5 × 10⁵ cells/mL. Briefly, the cells were centrifuged at 1,200 rpm for 5 min at 4°C in order to remove the culture medium, and the pellet was washed with 1 mL of PBS 1×. Then it was lysed with about 200 μL of the lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM NaF, and NP-40 1%) in the presence of protease inhibitors cocktail (PIC; Sigma-Aldrich) and 200 μM of phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich). At the end, the samples were centrifuged for 15 min at 13,000 rpm at 4°C. The antibodies used were TNF (Santa Cruz) and IL-6, IL-8, and IL-10 (Abcam); Erk (Santa Cruz) was used to normalize the results.
The protein quantification was performed following the method of Bredford. The Biorad protein assay was based on the change in the color of the dye Coomassie Brilliant Blue G-250 in response to various concentrations of protein. The reagent binds mainly to the aromatic amino acid residues or bases, such as arginine, as standard was used a solution of bovine serum albumin to a known concentration. The reading was carried out in a spectrophotometer at a wavelength of 595 nm as it develops a color tending toward blue, the intensity of which is directly proportional to the protein concentration present in the samples. Image J was used to quantify the expression of proteins.

Colon and liver tissues of experimental animals were homogenized using a 1 mL Radio-immunoprecipitation assay (RIPA, Invitrogen, Carlsbad, CA, USA) buffer, and proteins were separated via centrifugation at 13,000 rpm for 5 min at 4 °C. The separated proteins were quantified using the Bradford assay. The extracted proteins were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into a polyvinylidene fluoride (PVDF, Bio-Rad, Hercules, CA, USA) membrane. Nonspecific proteins were blocked with 5% skimmed milk containing phosphate-buffered saline with Tween 20 (PBST). After blocking, the PVDF membrane was washed thrice with PBST and once with PBS, and the primary antibody was reacted overnight at 4 °C. Then, the PVDF membrane was washed thrice with PBST and once with PBS, and the secondary antibody was reacted for 2 h at room temperature. For Bax, Bcl-2, caspase-3, caspase-9, p53, IL-6, NF-κB p65, IκB-α, TNF, and β-actin, Santa Cruz (Dallas, TX, USA) primary antibodies were used, and the proteins were detected using an Amersham Imager 680 (GE Healthcare Life Sciences, Chicago, IL, USA). The experiment was conducted at least three times.