Pgl4.14 reporter plasmid

To examine the effect of PB on the activation of the Il17 promoter, Jurkat cells were co-transfected with pCMV-β-Gal plasmid (Clontech, Mountain View, CA), pCMV10-3xFlag-RORγ plasmid, and a pGL4.14 reporter plasmid (Promega Corp., Madison, WI, USA) under the control of human Il17-3kb-CNS promoter^{46 (link)}, and then treated with vehicle, or PB (0.5μmol/l), or GW9662 (1.0 μmol/l) for 1 h before PB incubation. After 24 h, cell extracts were lysed and the firefly luciferase and β-galactocidase activities were measured using a Luciferase Assay Substrate kit (Promega) and a Luminescent β-galactosidase Detection kit II (Clontech), respectively.

PB was provided by professor Chen with extraction by 95% ethanol, column chromatography by EPE (ether: petroleum ether = 1:5) and recrystallization in methanol. The purity of PB was >98% determined by HPLC analysis^{30 (link)}. Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 medium, and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). PD, DMSO, DNFB (≥99% pure), monensin, ionomycin, phorbol-12-myristate-13-acetate (PMA) and GW9662 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-mouse CD4-FITC and IL-17A-PE-Cyanine7 antibodies were purchased from eBioscience (San Diego, CA). Anti-mouse IκBα, and phospho-IκBα (Ser32) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). CD3ε mAb, GAPDH and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Abcam Technology. Mouse interleukin (IL)-17, IL-22, IL-1β and tumor necrosis factor α (TNF-α) Enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems Inc. (Minneapolis, Minn., USA), and IgE ELISA kit was from Shibayagi Co. (Shibukawa, Japan). pRL-TK, pGL4.14 reporter plasmid, and Dual Luciferase II reporter assay kit were purchased from Promega (Promega Corp., Madison, WI, USA). Enhanced chemiluminescence (ECL) kit and bicinchoninic acid (BCA) protein assay kit were purchased from Pierce Biotechnology.