Evaluation of NF-κB/MAPK signaling pathway by western blotting

RAW264.7 cell proteins were extracted with RIPA lysis buffer (cat. no. C500007; Sangon Biotech Co., Ltd.) containing protease (cat. no. BL104A; BioSharp Life Sciences) and phosphatase inhibitor (cat. no. BL612A; BioSharp Life Sciences). Protein (40 µg/lane) was electrophoresed on a 12% acrylamide resolving gel and 5% stacking gel, then transferred to a PVDF membrane (EMD Millipore) for 30 min at 120 V. The membrane was blocked with 5% BSA (Biosharp Life Sciences) for 4 h at 26˚C and incubated with the appropriate diluted primary antibody at 4˚C for 16 h. Following five washes with TBS containing 0.05% Tween-20 (TBS-T), the membrane was incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (cat. nos. A0216 and A0208; 1:500). The membrane was washed in TBS-T thrice, visualized by chemiluminescence (cat. no. 32109; Thermo Fisher Scientific, Inc.) and the results were analyzed using Quantity One Software version 4.6.9 (Bio-Rad Laboratories, Inc.).