Wb 0071

At the end of cell culture, cells were washed twice with 0.9% NaCl at 4°C. The total protein in each well was extracted using 100 µl RIPA lysis buffer containing a protease inhibitor (WB-0071; Dingguo Changsheng Biotechnology Co., Ltd.) for 3 min on ice. Homogenates were centrifuged at 12,800 × g for 20 min at 4°C. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Shanghai Biyuntian Biotechnology Co., Ltd.). The supernatants were used for western blot analysis. Proteins (50 µg) were separated via 12% SDS-PAGE, transferred to nitrocellulose membranes, blocked in 5% skimmed milk at 37°C for 1 h, and incubated overnight at 4°C with anti-LC3 (1:3,000), anti-caspase-12 (1:1,000), anti-PERK (1:2,000), anti-p-PERK (1:2,000), anti-ATF4 (1:1,000), anti-CHOP (1:1,000), anti-IRE1 (1:3,000), anti-p-IRE1 (1:3,000), anti-TRAF2 (1:1,000), anti-Beclin-1 (1:1,000) and anti-β-actin (1:5,000) primary antibodies. The membranes were then incubated for 2 h at room temperature with appropriate secondary antibody (Goat Anti-Rabbit IgG, 1:5,000). The blots were visualized using an enhanced chemiluminescence detection system (Cell Signaling Technology, Inc.). Images were processed using Quantity One software (version 4.6; Bio-Rad Laboratories).

Total protein was extracted from the colon tissue samples via radio immunoprecipitation assay (RIPA) lysis buffer (WB-0071; Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). Protein samples were mixed with sample buffer (5X) and denatured by boiling. For immunoblots, proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis on a 10% polyacrylamide gel. Samples were electrophoresed at 80–100 V for 2 h until the dye migrated to the bottom of the gel. The proteins were transferred to polyvinylidene difluoride membranes and the membranes were blocked in blocking buffer (5% non-fat dry milk) for 2 h at room temperature, and incubated with primary antibodies against CRF-R1 (ab59023; 1:500; Abcam, Cambridge, MA, USA) or p-p65 NF-_ΚB (3031; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) and β-actin (wl01845; 1:1,000; Wanlei Biotechnology, Co., Ltd., Shenyang, China) overnight at 4°C. Membranes were then incubated with a horseradish peroxidase (HRP)-conjugated anti-goat (SA00001-4; 1:7,000; Proteintech Group, Inc., Rosemont, IL, USA) and anti-rabbit (Wla023a; 1:8,000; Wanlei Biotechnology, Co., Ltd.) secondary antibody for 1 h at room temperature. The proteins were visualized with a chemiluminescent substrate (WBULS0100; EMD Millipore, Billerica, MA, USA) using an imaging system (Bio-Rad Laboratories Inc., Hercules, CA, USA).