Ripa assay

Colon tissue samples were acquired and lyzed using RIPA assay (Beyotime Institute of Biotechnology, Haimen, China). Protein was quantified using a BCA kit (Beyotime Institute of Biotechnology). A total of 10 µg protein was used per sample to measure the levels of IL-22 (cat. no EH027-96; ExCell Bio, Shanghai, China) and IL-22BP (cat. no. EK2506; Nanjing Senberga Biotechnology Co., Ltd., Nanjing, China) using ELISA kits.

Brain samples were transferred into ice-cold RIPA assay (Beyotime Institute of Biotechnology; Shanghai, China) containing a cocktail of protein phosphatase and proteinase inhibitors. Samples were collected at 15,000 × g at 4°C for 10 min. The supernatant was assayed using a Bio-Rad Bradford Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein (50 µg) was separated using 8–10% SDS-PAGE and blots were blocked in buffer solution containing 5% milk and 0.1% Tween-20 in PBS, following transfer onto polyvinylidene fluoride membranes. Membranes was then blotted for 2 h at room temperature with antibodies targeting inducible nitric oxide synthase (iNOS; 1:4,000; Sigma-Aldrich; Merck KGaA), Nrf2 (1:4,000; Sigma-Aldrich; Merck KGaA), HO-1 (1:4,000; Sigma-Aldrich; Merck KGaA) or β-actin (1:4,000; Sigma-Aldrich; Merck KGaA) at 4°C overnight. Membranes were washed with TBST for 15 min and incubated with goat anti-rabbit HRP secondary antibody (1:3,000; HA-1001-100; Hangzhou Hua'An Biotechnology Co., Ltd., Hangzhou, China) for 1 h at 37°C. Protein expression was detected using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology, Shanghai, China) and quantified using Bio-Rad Image Lab 3.0 (Bio-Rad).