Dnp hsa

RPMI1640, fetal bovine serum (FBS), and the enhanced chemiluminescence (ECL) Western blot detection reagent were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Mouse anti-dinitrophenyl (DNP) IgE was purchased from Sigma Chemicals (St. Louis, MO, USA). DNP-HSA was from Biosearch Technologies (Petaluma, CA, USA). The antibodies specific for phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-PLCγ, phospho-IκB, IκB, phospho-IKKα/β, β-actin, and the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibodies specific for phospho-cPLA₂, NF-κB p65, lamin B, Lyn, Fyn, and Syk, as well as Bay 61-3606 reagent were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The LTC₄ enzyme linked immunoassay (EIA) kit, and the antibody for COX-2 were from Cayman Chemical (Ann Arbor, MI, USA). Histamine enzyme linked immunosorbent assay (ELISA) kit was purchased from Demeditec Diagnostics GmbH (Kiel, Germany). AB23A was obtained from Push Bio-technology Co., Ltd (Chengdu, China), of which the purity is ≥98%.

β-Hexosaminidase (β-hex) release, as a marker of MCs degranulation, was assayed by using a fluorometric assay. The RBL-2H3 cells were plated into 12-well plates (9 × 104 cells per well) and BMMCs were plated into 96-well plates (8 × 103 cells per well). After sensitized with 0.1 µg/mL DNP (dinitrophenylated)-specific IgE (Sigma, USA) overnight, the cells were then treated with or without PC (1, 10, or 100 nmol/mL, P7443, Sigma-Aldrich, USA) for 12 h. Then the PC-containing medium was removed and washed cells with dulbecco’s phosphate buffered saline(D-PBS) twice. To examine degranulation, the cells were stimulated with 1 µg/mL DNP-HSA (dinitrophenylated human serum albumin, Biosearch Technologies, USA) for 30 minutes. Then, cell culture supernatant was collected, and cell pellets were lysed with NP-40 (3 mM). Supernatants and cell lysates, respectively, were incubated with reaction buffer (3 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide, pH 4.4, Sigma, USA) for 1.5 h at 37 °C. The mixture reaction was terminated by 150 µL stop solutions (Na2CO3/NaHCO3, pH 10.6). Then, absorbance at 405 nm was measured with a microplate reader (BIO-RAD Laboratories, USA). The β-hex release was evaluated by using the following formula: β-hex release(%)= [(absorbance of the supernatant)/(optical absorbance of the supernatant+ absorbance of the cell pellet)]×100%.