Phosphorylated mkk4

Orobol was provided by Prof. Byung-Gee Kim (Seoul National University, Korea). Orobol was o-hydroxylated by Genistein with a > 99.9% conversion yield. It underwent biotransformation by tyrosinase expressed in Bacillus megaterium. Genistein was purchased from Sigma-Aldrich (USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Wel-Gene (Republic of Korea). Fetal bovine serum (FBS) was purchased from Sigma-Aldrich and penicillin/streptomycin solution from Mediatech, Inc. (USA). Antibodies against phosphorylated IKKα/β, total IKKα, total IKKβ, phosphorylated IκBα, total IκBα, phosphorylated mitogen- activated protein kinase kinase (MEK)1/2 (Ser217/Ser332), total MEK1/2, phosphorylated dual-specificity mitogen- activated protein kinase kinase (MKK)3/6 (Ser189/Ser207), total MKK3β, phosphorylated MKK4, total MKK4, phosphorylated p38, total p38, phosphorylated c-Jun N-terminal kinase (JNK), total JNK, phosphorylated p90 ribosomal s6 kinase (RSK), total RSK, phosphorylated mitogen- and stress-activated protein kinase 1 (MSK1) (Ser360), and total MSK1 were purchased from Cell Signaling Technology Inc. (USA). Antibodies against phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) (Thr202/Tyr204), total ERK1/2, and β-actin were purchased from Santa Cruz Biotechnology (USA). Protein assay kits were obtained from Bio- Rad Laboratories (USA).

Primary antibodies against β-actin, JNK, ERK, and p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The iNOS, COX-2, and phosphorylated-IκB-α, phosphorylated-JNK, phosphorylated-ERK, phosphorylated-p38, and phosphorylated-MKK4 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). A protein assay kit was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Orientin, isoOrientin, vitexin, isovitexin, rutin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), LPS, interferon- (IFN-) γ, and Griess reagent were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Welgene Inc. (Daegu, Republic of Korea). All solvents used were of HPLC grade.