Anti erk antibody

Anti-LAT, anti-PLC-γ1, and anti-Erk antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-NTAL NAP-07 monoclonal antibody was from EXBIO (Prague, Czech Republic). Antibodies binding phospho-Erk, phospho-PLC-γ1-Tyr783, ZAP70, phospho-ZAP70-Tyr319, phospho-MEK-Ser221, and anti-MEK were from Cell Signaling Technology. Anti-β-actin, anti-CD3 (OKT3), and anti-CD4 monoclonal antibodies were provided by Biolegend (San Diego, CA, USA).

The following primary antibodies were used in this study: mouse monoclonal anti-myc antibody (1:1000 for WB; Cell Signaling Technology, Inc., Denver, MA, USA); rabbit polyclonal anti-Erk antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies used were: Horseradish peroxidase-conjugated goat anti-mouse antibodies (1:5000 for WB; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:10,000 for WB; Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Recombinant human visfatin was purchased from PeproTech (Rocky Hill, NJ, USA). The tyrosine kinase inhibitor of Janus kinase 2 (JAK2), AG490, was from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The NF-κB inhibitor Bay 7082 was purchased from Calbiochem (San Diego, CA, USA). Antibodies against phospho-insulin receptor (IR), total IR, insulin receptor substrate 1 (IRS-1 (Thy612)), phospho-Glycogen Synthase Kinase 3 α/β (GSK-3α/β), protein kinase B (AKT), phospho-AKT, JAK2, phospho-JAK2, signal transducer and activator of transcription 3 (STAT3), phospho-STAT3, IκB kinase (IKK), phospho-IKKα/β, inhibitor of kappa B (IκBα), NF-κB, phospho-NF-κB, c-Jun N-terminal kinases (JNK), phospho-JNK, and p-38 mitogen-activated protein kinases (p-38) and phospho-p-38 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-GSK-3α/β, anti-actin, anti-phosphor-extracellular signal-regulated kinase (ERK), and anti-ERK antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti IRS-1 (Ser307) antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY, USA). Culture media, culture supplements, and fetal bovine serum (FBS) were obtained from Gibco-BRL (Grand Island, NY, USA).

Stimulatory antibodies against human CD3 and CD28 for the stimulation of T cells were obtained from BioXcell (West Lebanon, NH, USA). Recombinant human TNFα was purchased from PeproTech EC Ltd. (London, UK). MTT powder (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), TRIZOL reagent and radioimmunoprecipitation assay (RIPA) buffer for Western blot analysis, phorbol 12-myristate 13-acetate (PMA) and A23187 were provided by Sigma Chemical Co. (St. Louis, MO, USA). Staphylococcus aureus enterotoxin E (SEE) was obtained from Toxin Technology (Sarasota, FL, USA). ECL Western blotting detection reagents and an apoptosis AnnexinV/PI assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). SYBR Premix Ex Taq was provided by TaKaRa (Shiga, Japan). Anti-IL-2 antibodies, anti-β-actin and anti-ERK antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against p65, LaminB, IκBα, phosphorylated IκBα, phosphorylated ERK, phosphorylated p38, p38, phosphorylated JNK and JNK were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-CD69 conjugated with APC were provided by eBiosciences. The RT PreMix kit was obtained from Enzynomics (Daejeon, Korea). DSS (molecular weight: 36,000–50,000 Da) was purchased from MP Biomedicals (Irvine, CA, USA).