Anti ant2

THP-1 monocytes were seeded in 6-well plates at a density of 4.5 × 10⁶ per well and incubated with 200 nm phorbol 12-myristate 13-acetate (PMA) for 72 h. Afterwards, the medium was changed to RPMI/FBS without PMA for 24 h. The cells were infected with Mtb Erdman at an MOI of 2 and supernatants were collected 24 h post infection. The supernatants were precipitated using methanol and chloroform and resuspended in Laemmli buffer. Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis as previously described. The following antibodies were used: anti-ANT2 (14671; RRID: AB_2798562), anti-β-actin (4970), anti-cleaved IL-1β (83186; RRID: AB_2800010), and anti-NLRP3 (15101; RRID: AB_2722591) from Cell Signaling Technology and anti-GSDMD (HPA044487; RRID: AB_2678957) from Merck.

Whole cell lysate was prepared with lysis buffer (50 mM Tris pH = 7.5, 0.2 M NaCl, 1% Tween-20, 1% NP40, 1 mM sodium orthovanadate, 2 mM β-glycerophosphate and prote-ase inhibitors). Twenty μg of protein were seperated by 10% SDS polyacrylamide gel electrophoresis (PAGE) and transferred to pol-yvinylidene difluoride membranes (Merck, Darmstadt, Germany). Membranes were blocked by 5% BSA in Tris-buffered saline (20 mM Tris, pH 7.4, 150 mM NaCl) containing 0.1% Tween-20 (TBST) and incubated with primary antibodies overnight at 4 °C. Membranes were then reacted with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, Dallas, TX, USA) and imaged by X-ray films. Primary antibodies for western blot were as follows: anti-ANT1 (NBP83928, Novus, St Charles, MO, USA), anti-ANT2 (14671, Cell Signaling, Danvers, MA, USA), anti-GPX1 (3286S, Cell Signaling), anti-SOD1 (2770S, Cell Signaling), an-ti-SOD2 (13141S, Cell Signaling), anti-Actin (sc-8432, Santa Cruz), and anti-Ku70 (SC-17789, Santa Cruz).

All chemicals were purchased from Sigma (St. Louis, MO, USA). For immunoblotting, the following antibodies were used: anti-ANT2, anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), anti-Tom20, anti-cytochrome C, anti-53BP1 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-ATP5B (Sigma, St. Louis, MO, USA), anti-γH2AX (Millipore, Billerica, MA, USA), anti-NDUFA9, anti-SDHA, anti-Cox5a, anti-UQCRC2, anti-ANT1 and anti-VDAC (Abcam, Cambridge, UK). All antibodies were diluted 1:1000 in 2.5% non-fat milk. Horseradish peroxidase (HRP) conjugated β-actin (ThermoFisher, Waltham, MA, USA) was used as a loading control. IgG-HRP anti-rabbit (170-6515) and anti-mouse (170-6516) secondary antibodies produced in goat were purchased from BioRad Laboratories (Hercules, CA, USA). Secondary antibodies were diluted 1:10,000 in 2.5% non-fat milk.