Bronchoalveolar Lavage Fluid (BALF) Collection and Western Blot Analysis

Mice infected by recombinant pMV262/MS, Rv0888NS/MS, D438A/MS, and H481N/MS were anesthetized with diethyl ether, and the trachea was exposed. Subsequently, a closed IV catheter system (BD Intima, Franklin Lakes, NJ, USA) was inserted into the trachea and washed with 500 µl PBS three times. The collected BALF was incubated with 100 U DNase I (Amresco, Solon, OH, USA) at 37°C for 30 min, and the supernatant was analyzed by Western blotting with the following primary antibodies: anti-histone H4 (Abcam, ab17036), anti-histone H3 (citrulline R2 + R8 + R17) (Abcam), and anti-MPO (Abcam, ab139748). Then, incubation with secondary anti-mouse IgG Dylight 680-labeled antibody or anti-rabbit IgG Dylight 800-labeled antibody (KPL, USA) was carried out. Reactivity was detected and recorded on a Li-Cor Odyssey imaging system (Li-Cor Biosciences, USA).