4 nitrophenyl n acetyl β d glucosaminide p nag

The coding DNA of full-length Ara h 1 [28 (link)], adding 6 × his tag, was cloned into a pET-32a expression vector by Shenggong biology company (Shanghai, China). Recombinant Ara h 1 (rAra h 1) was produced by Escherichia coli BL21(DE3) pLysS and purified as described [17 (link)]. Bovine serum albumin (BSA), microbial transglutaminase (MTG), 3,3′,5,5′-tetramethylbenzidine (TMB), isopropyl-β-D-thiogalactopyranoside (IPTG), 4-nitrophenyl-N-acetyl-β-D-glucosaminide (PNAG), 1-anilino-8-naphthalene-sulfonate (ANS), and peroxidase (HRP)-labeled goat anti-human IgE were purchased from Sigma Chemical Co. (St Louis, MO, USA). Precast 4–20% electrophoresis gel kits, loading buffer, DL-dithiothreitol (DTT), BCA Protein Assay Kit, and peroxidase (HRP)-labeled goat anti-rabbit IgG were obtained from Solarbio Co. (Beijing, China). The standard protein marker was obtained from TransGen Biotech Company (Beijing, China). Rabbit anti-Ara h 1 antibody was kindly provided by the university of Manchester. Enhanced chemiluminescence (ECL) kit for immunoblotting and ImmunoCAP assay kit were purchased from Beyotime Co. (Shanghai, China).

Granule release was measured by detection of β-hexosaminidase in the cell culture supernatant as previously described (23 ). Briefly, 5 × 10⁴ cells in Tyrodes solution supplemented with 10 mM HEPES and 0.04% bovine serum albumin were added to 96-well plates. Cells were infected with the indicated bacterial strains at an MOI of 10 for 90 min at 37°C. After incubation, the plate was centrifuged to collect supernatants containing released granules and supernatants transferred to a new 96-well plate. Remaining cells were lysed with 0.05% Triton X-100 to assess granules remaining within the cells. Supernatants and lysates were incubated with 3.5 mg/mL 4-nitrophenyl N-acetyl-β-D-glucosaminide (PNAG, Sigma) in citrate buffer (200 mM citric acid and 100 mM Na₂HPO₄·7H₂O, pH = 4.5) for 90 min. at 37°C. Reactions were stopped with 100 mM glycine (pH = 10.7) and plate absorbance was read at 405 nm.

Eagles’ Minimum Essential Medium (EMEM) was purchased from American Type Culture Collection (ATCC) (Manassas, VA, U.S.A.). Fetal Bovine Serum (FBS), antibiotics (penicillin and streptomycin) and trypsin were purchased from Gibco (Rockford, IL, U.S.A.). 4-Nitrophenyl N-acetyl-β-D-glucosaminide (PNAG) was purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). The ELISA kit for TNF-α was purchased from R&D systems (Minneapolis, MN, U.S.A.). Ketotifen fumarate was purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). NP-40 surfact-amps detergent was purchased from Thermo Scientific (Waltham, MA, U.S.A.). Carboxymethyl cellulose (CMC) was purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Anti-dengue IgG (pan) mouse antibody was purchased from Acris (Herford, Germany). Horse-radish peroxidase (HRP) was purchased from BioLegend (GmbH, Germany), metal enhanced 3′-diaminobenzidine (DAB) peroxidase substrate was purchased from Thermo Scientific (Pierce, Rockford, IL, U.S.A.).

Mouse TSLP was purchased from ProSpec (catalog #CYT-1135, East Brunswick, NJ, USA). GSK1016790A (catalog #G0798), HC067047 (catalog #616521), carvacrol (catalog #282197), 2-Aminoethoxydiphenylborane (2-APB, catalog #100065), SLIGRL-NH₂ (SLIGRL, catalog #S9317), FSLLRY-NH₂ (FSLLRY, catalog #SML0714), HC030031 (catalog #H4415), capsazepine (catalog #C191), baicalein (catalog #465119), dispase II (catalog #D4693), 4-nitrophenyl N-acetyl-β-D-glucosaminide (pNAG, catalog #N9376), and toluidine blue (catalog #89640) were purchased from Sigma-Aldrich (St, Louis, Missouri, USA).

3-(4,5-dimethylthiazol-2-yl)2-,5-diphenyltetrazolium bromide (MTT), dinitrophenyl-human serum albumin (DNP-HSA), anti-DNP-IgE and 4-nitrophenyl N-acetyl-β-d-glucosaminide (p-NAG) were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Antibodies against phospho-protein kinase B (Akt; #9271), phospho-cPLA₂ (#2831), phospho-extracellular signal-regulated kinase 1/2 (ERK; #9101), phospho-c-Jun N terminal kinase 1/2 (JNK; #9251), phospho-Src family protein kinase (Lyn; #2731), phospho-p38 (#9211), phospho-PKCδ (#2055), phospho-PLCγ1 (#2821), phospho-spleen tyrosine kinase (Syk; #2710), horseradish peroxidase-linked anti-rabbit secondary antibody (#7074) and anti-mouse secondary antibody (#7076) were purchased from Cell Signaling Technology (Beverly, MA, USA). Specific antibodies against phospho-Fyn (orb128087) were obtained from Biorbyt Ltd. (Cambridge, UK). A specific antibody against 5-LO (#10007820) and EIA kits for LTC₄, LTB₄ and PGE₂ were obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). ELISA kits for IL-4 and TNF-α were purchased from R&D systems, Inc. (Minneapolis, MN, USA). Minimum Eagle’s medium (MEM) and fetal bovine serum (FBS) were from the American Type Culture Collection (Manassas, VA, USA). All other chemicals and reagents used were of analytical grade.