RAW264.7 cells were seeded at a density of 8×105 cells/well in a 6-well plate, and were then pretreated with or without specific inhibitors (25 μM SB203580, 25 μM U0126 or 20 μM SP600125) for 2 h at 37°C, followed by incubation with WCCP-A-b (200 μg/ml) at 37°C for 24 h. Western blot analysis was performed as described by Meng et al (36). RAW264.7 cells were rinsed twice with cold PBS and lysed in lysis buffer (50 mM Tris/acetate, pH 7.4, 1 mM EDTA, 0.5% Triton X-100, 150 mM sodium chloride, 0.1 mM PMSF and Roche incomplete protease inhibitor cocktail). Protein concentration was measured using the Bradford method. Equal amounts of protein (30 μg/lane) were separated via 12% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked in 3% BSA at room temperature for 1 h and then blotted with specific antibodies, and proteins were detected using an electrochemiluminescence reagent (Tanon Science and Technology Co., Ltd.). Both the primary and secondary antibody incubations were 1 h at room temperature. Primary antibodies (1:1,000) against phosphorylated (p)-JNK (cat. no. 4668s), JNK (cat. no. 9252s), p-ERK (cat. no. 9101s), ERK (cat. no. 9102s), p-p38 (cat. no. 9215s) and p38 (cat. no. 9212s) were obtained from Cell Signaling Technology, Inc. The antibody against β-actin (cat. no. 612657) was purchased from BD Biosciences. HRP-conjugated goat anti-rabbit IgG (cat. no. AS014) and goat anti-mouse IgG (cat. no. AS003) secondary antibodies (1:5,000) were obtained from ABclonal Biotech Co., Ltd.

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