For Western blot analysis, prepared WCL and nuclear fraction were denatured at 95°C for 10 min, separated by 12.5% SDS-PAGE gel. Proteins were transferred onto nitrocellulose membranes and blotted with rabbit anti-IRF3 (cat. A303-384A; Bethyl Laboratories Inc.), anti-IRF7 (cat. 3941; Prosci Inc.), anti-TBK1 (cat. 3504; Cell Signaling Technology), anti–phospho-TBK1 (Ser172; cat. 5483; Cell Signaling Technology), anti–RIG-I (cat. 3743; Cell Signaling Technology), anti–MDA-5 (cat. 5321; Cell Signaling Technology), anti-cGAS (cat. Sc-515777; SCBT), anti–β-actin (cat. 4970; Cell Signaling Technology), anti-MAVS (cat. Sc-365333; SCBT), anti-STING (cat. NBP2-24683SS), and anti-histone H3 (cat. 9717; Cell Signaling Technology) antibodies, followed by goat anti-rabbit IgG-HRP (cat. 31460; Thermo Scientific).
For M.tb culture supernatant proteins, M.tb strains were grown in Sauton’s liquid medium until midexponential phase, and culture supernatant fraction was prepared from 50 ml bacterial culture as described previously (Reyna et al., 2016 (link)). 10 µg of sample was loaded into 12% SDS-PAGE gel, and M.tb RNA polymerase subunit β (RNAP-β) and EsxB (CFP-10) proteins were probed using mouse anti–RNAP-β antibody (ab12087; Abcam) and rabbit anti-EsxB antibody (NR-13801; BEI Resources), respectively.
For M.tb culture supernatant proteins, M.tb strains were grown in Sauton’s liquid medium until midexponential phase, and culture supernatant fraction was prepared from 50 ml bacterial culture as described previously (Reyna et al., 2016 (link)). 10 µg of sample was loaded into 12% SDS-PAGE gel, and M.tb RNA polymerase subunit β (RNAP-β) and EsxB (CFP-10) proteins were probed using mouse anti–RNAP-β antibody (ab12087; Abcam) and rabbit anti-EsxB antibody (NR-13801; BEI Resources), respectively.
