P rsk

Eriocitrin, narirutin, naringin, neohesperidin, hesperidin, and synephrine were purchased from the National Institutes for Food and Drug Control (Beijing, China). LPS and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against ERK, p-ERK, JNK, p-JNK, p38, p-p38, p-AMPKα, ACC, p-ACC, p65, p-p65, RSK, p-RSK, MSK, p-MSK, α-Tubulin and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against AMPKα was purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against IκBα and p-IκBα were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against MPO was purchased from abcam (Cambridge, MA, USA). Horseradish peroxidase-conjugated secondary antibody was purchased from Hangzhou Baoke Biotechnology. Co., Ltd. (Hangzhou, China). Alexa Fluor 488-conjugated AffiniPure donkey anti-rabbit IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Dexamethasone (Dex) was purchased from North China Pharmaceutical Qinhuangdao Co., Ltd. (Hebei, China). Mouse Inflammation Kit and IL-1β enzyme-linked immunosorbent assay (ELISA) Kit were purchased from BDBiosciences (San Jose, CA, USA). Enhanced BCA Protein Assay Kit and Enhanced Chemiluminescence (ECL) Kit were purchased from Beyotime Biotechnology (Shanghai, China).

All melanoma cells were washed with ice-cold phosphate buffered saline (PBS) twice and lysed with RIPA buffer containing phosphatase and protease inhibitors (all from Sigma Aldrich, St. Louis, MO). Protein extracts were separated with SDS-PAGE in 4-12% tris-glycine gels and transferred to immun-blot PVDF membrane. After blocking for 1 hour in PBS containing 0.1% Tween 20 and 5% nonfat milk or 5% bovine serum albumin (BSA) in PBS, the membrane was exposed to various primary antibodies overnight, followed by secondary antibodies conjugated to horseradish peroxidase. ECL-Plus kit (Amersham Biosciences Co, Piscataway, NJ) was used to check immunoreactivity and blots were scanned using a Typhoon scanner (Amersham Biosciences Co.). Primary antibodies included pERK Thr202/Tyr204, total ERK, pMEK Ser217/221, total MEK, pAKT Ser473, total AKT, pRSK, total RSK, beta-actin (all from Cell Signaling Technology, Danvers, MA).