P nf κb

Total proteins were extracted using RIPA buffer (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The protein lysate was subjected to electrophoresis and transferred onto a PVDF membrane (Bio-Rad, CA, United States). The following primary antibodies sourced from CST (Shanghai, China): p-MEK (Ser217/221; 1:1000; 9154), MEK (1:1000; 8727), p-ERK1/2 (Thr202/Tyr204; 1:2000; 4370), and ERK (1:1000; 4695), PD-1 (1:1000; 84651), PD-L1 (1:1000; 60475), and p-NF-κB (1:1000; bs-0982R) from Bioss (Beijing, China), NF-κB (1:1000; sc-8008) from Santa Cruz Biotechnology (Texas, United States) were used to incubate the membrane, respectively. The proteins were detected using ECL Plus Reagent (Beyotime) and quantified using Image Lab software.

Antibodies against NF-κB (Bioss, China), p-NF-κB (Bioss, China), and β-actin (Proteintech, USA) were used in this study. Briefly, frozen intestinal samples were homogenized and subsequently determined for protein concentration using the bicinchoninic acid (BCA) protein assay kit (Wellbio, China). Equal quantity of protein samples was separated on SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% skim milk for 1 h at room temperature, the membranes were incubated with the indicated primary antibodies at 4°C overnight. Then, blots were stripped, followed by the incubation with HRP-conjugated secondary antibody (Proteintech, USA) for 1 h. The chemiluminescence signals were obtained using ECL Plus kits (Thermo, USA). The band density was normalized to β-actin and expressed as a relative level to control value.