Total msk1

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).

Antibodies against total ERK1/2 (#9102), phospho-ERK1/2 (Thr202/Tyr204; #9101), total IκBα (#9242), total JNK1/2 (#9252), phospho-p38MAPK (Thr180-Tyr182; #9211), total MK2 (#3042), phospho-MK2 (Thr334; #3042), total MSK1 (#3489), phospho-MSK1 (Thr581; #9595), phospho-c-Jun (Ser73; #9164) and phospho-IKKα/β (Ser176/180; #2697) were purchased from Cell Signaling Technology. Antibodies to Phospho-CREB (Ser133, #06-519) was from Millipore, anti-p38α (#sc-535) and anti-DUSP1 (#sc-373841) were from Santa Cruz, anti-active phospho-JNK1/2 (Thr183-Tyr185; #MAB1205) from R&D System, and anti-α-Tubulin (#T9026) from Sigma. Secondary antibodies from Invitrogen (Waltham, Massachusetts, United States) included Alexa Fluor 680 donkey α-sheep IgG (H + L) (#A21102), Alexa Fluor 680 goat α-rabbit IgG (H + L) (#A21109), Alexa Fluor 700 goat α-mouse IgG (H + L) (#A21036).
Kinase inhibitors SB203580 (inhibits p38α/p38β (Kuma et al., 2005 (link))) was purchased from Selleckchem, and SB747651A (inhibits MSK1, (Naqvi et al., 2012 (link))) from Axon Medchem. PD184352 (MKK1 inhibitor, (Kuma et al., 2005 (link))) and BIRB0796 (p38α/p38β/p38γ and p38δ inhibitor, (Kuma et al., 2005 (link))) were from the Division of Signal Transduction Therapy (DSTT); University of Dundee (Dundee, UK). JNK-IN-8 (JNK inhibitor (Zhang et al., 2012 (link))) was from Calbiochem.