Detection of Inflammatory Proteins by Western Blot

RAW 264.7 cells were treated with LPS (1 μg/ml) alone or together with PBD-2 (10 μg/mL). After 6 h, the cells were lysed, and inflammatory proteins were detected through Western blot. Proteins were firstly analyzed with SDS-PAGE and then electro-transferred onto a PVDF (#ISEQ00010, Biosharp^®, Beijing, China) membrane. The PVDF membrane was blocked with 1/100 bovine serum albumin (BSA, #V900933-100G, VETEC, Sigma-Aldrich^®, St. Louis, MO, USA) at 10 μg/ml at 37°C for 2 h, washed with PBST five times, incubated with the antisera against p-p65 (# 3033), p65 (# 4764), p-ERK (# 4370), ERK (# 4695), p-p38 (# 4511), p38 (# 8690), p-JNK (# 4668), JNK (# 9258) (Cell Signaling Technology^®, Danvers, MA, USA), and β-actin (# AA128, Beyotime^®, Shanghai, China) at 2 μg/ml at 37°C for 2 h and washed five times again. The PVDF membrane was subsequently incubated with 1 μg/ml secondary antibody, namely, goat anti-rabbit IgG (# 7074S, Cell Signaling Technology^®, Danvers, MA, USA), at 37°C for 2 h and washed five times. Lastly, immune-reactive bands were detected using an ECF Western blot system (Tanon 5200 Multi, Shanghai, China) and quantified using AlphaImager 2200 analysis software.