1 cyano 4 dimethylaminopyridinium tetrafluoroborate cdap

M. bovis BCG Pasteur and Mtb H37Rv were grown in minimal medium (MM) [KH₂PO₄ 1 g/l, Na₂HPO₄ 2.5 g/l, asparagine 0.5 g/l, ferric ammonium citrate 50 mg/l, MgSO₄×7 H₂O 0.5 g/l, CaCl₂ 0.5 mg/l, ZnSO₄ 0.1 mg/l, 0.1% (v/v) glycerol, and with or without Tyloxapol 0.05% (v/v; Sigma), pH 7.0] or in Middlebrook 7H9 supplemented with 10% (v/v) OADC enrichment (Becton Dickinson Microbiology Systems, Spark, MD), 0.5% (v/v) glycerol and with or without Tyloxapol 0.05% (v/v) for 14 days in a 5% CO₂ incubator at 37°C. Mtb lineages were a gift from Sebastien Gagneux. Mtb lineages were systematically grown in MM supplemented with 30 mM pyruvate.
Recombinant Ag85b was obtained from AERAS Tb vaccine Foundation (Rockville, MD). Recombinant PA (Protective Antigen from Bacillus anthracis) was obtained from David Axelrod Institute, Albany, NY). The 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) and the other reagents used during the conjugation reaction were purchased from Sigma. The CS-35 monoclonal antibody recognizing LAM and AM, was obtained from BEI resources (Manassas, VA). The monoclonal antibody 9d8 specifically recognizes mycobacterial capsular AM [10 (link), 54 (link)]. The monoclonal antibody 24c5 specifically recognizes mycobacterial capsular α-glucan [55 (link)]. Alhydrogel was purchased from InvivoGen (San Diego, US).

Dulbecco's modified Eagle's medium was purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum was from Sijiqing (Hangzhou, Zhejiang, China). Penicillin, streptomycin, and nuclear and cytoplasmic extraction kit were from Shenggong (Shanghai, China). Abs to phospho-IκB-α, IκB-α, NF-κB-p65, β-actin, and JSH-23 were all from Santa Cruz (Santa Cruz, CA, USA). PDTC, MG-132, BAY 11-7082, BCA protein assay kit, nitric-oxide synthase assay kit, and abs to histone H3 were from Beyotime (Nantong, Jiangsu, China). Abs specific to mouse TLR4 was from eBioscience (San Diego, CA, USA). LPS, o-phenylenediamine, fluoresceinamine (FLA), and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) were from Sigma-Aldrich (St. Louis, MO, USA). Enhanced chemiluminescence (ECL) substrate was from Thermo Scientific.