P nf κb p65

Frozen colon tissue samples were homogenized in lysis buffer to obtain total protein. Protein concentration was determined in supernatants using the BCA Protein concentration determination kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by SDS-PAGE and transferred to the polyvinylidene difluoride membrane. The membranes were blocked in 5% nonfat milk for 1 h, and membranes were incubated at 4°C overnight with primary antibodies to p-PERK, eIF2α, p-eIF2α, CHOP, NF-κB, β-actin (from Cell Signaling, Danvers, MA, USA), GRP78 (from Proteintech, Wuhan, Hubei, China), ATF4 (from Santa Cruz, Dallas, TX, USA), and p-NF-κB p65 (from ABclonal, Wuhan, Hubei, China). The membranes were incubated with secondary antibodies, anti-rabbit IgG or anti-mouse IgG (from Cell Signaling, Danvers, MA, USA) for 1 h at room temperature. Protein bands were quantified using ImageJ and the grey value of the targets was normalized by β-actin.

Total protein was extracted from RAW264.7 macrophages and primary peritoneal macrophages using radio immunoprecipitation lysis buffer (RIPA, Solarbio, Beijing, China) supplemented with phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitors (Solarbio, Beijing, China). The protein was collected, and its concentration was measured using a Pierce BCA protein assay kit (Thermo, Rockford, IL, USA). Equal amounts of proteins were separated by electrophoresis on SDS-PAGE gels and subsequently transferred onto PVDF membranes. Blocking was with TBST containing non-fat milk powder, the membranes were incubated overnight at 4 °C with primary antibodies. The primary antibodies against NRF2, p-AKT, AKT, iNOS (Cell Signaling Technology, Danvers, MA, USA), and HO-1, NQO-1, COX-2, p-GSK-3β, GSK-3β, p-AMPK-α1, AMPK-α1, p-ERK, EKR, p-JNK, JNK, p-P38, P38, p-NF-κB p65, NF-κB p65, and GAPDH (ABclonal, Wuhan, China) were diluted to a ratio of 1:2000. Before adding secondary antibodies (goat anti-rabbit or goat anti-mouse) diluted to a concentration of 1:10,000 (ABclonal, Wuhan, China), the membranes were washed four times with TBST for 15 min each time. Membranes were visualized using an Amersham Imager 600 (a gel imaging system from GE Co., Fairfield, CT, USA) after applying enhanced chemiluminescence. For detailed information of WB procedures, refer to our previous study [50 (link)].