Anti mda5

Protein extraction was performed by lysing the cells in Laemmli sample buffer and separated by SDS-PAGE using 7.5% polyacrylamide gels and electrotransferred to nitrocellulose membranes (Bio-Rad, Cat. No. 162-0115). Non-specific binding sites were blocked with 5% non-fat dry milk diluted in TBS Tween buffer (50 mM Tris, 0.5 M NaCl, 0.05% Tween-20, pH 7.4). The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778). The bound antibodies were labeled with anti-mouse (Bio-Rad, Cat. No. 1721011), anti-rat (Bio-Rad, Cat. No. 5204-2504) or anti-rabbit (GE Healthcare, Cat. No. NA934) horseradish peroxidase-conjugated secondary antibodies and were visualized by the ECL system using SuperSignal West Pico or Femto chemiluminescent substrates (Thermo Scientific, Rockford, IL, USA, Cat. No. 34580 and 34095) and X-ray film exposure. Densitometric analysis of immunoreactive bands was performed using Image Studio Lite Software version 5.2 (LI-COR Biosciences, Lincoln, Nebraska USA).

Cultured epithelial cells after stimulation were rinsed with DPBS and proteins were extracted using radio-immunoprecipitation assay (RIPA) buffer (GenDEPOT). Denatured proteins (25 μg) were fractionated on SDS-PAGE and the gels were transferred onto a polyvinylidene difluoride membrane (Bio-Rad, MA, USA). Thereafter the membranes were rinsed and probed with the primary antibodies in the refrigerator overnight at 4 °C; the primary antibodies employed for Western blots were anti-β actin (1:5000) which was obtained from Santa Cruz, USA, anti-viperin, anti-OAS, anti-Mx, anti-TLR3, anti-RIG1, anti-MDA5, anti-SOD1, anti-SOD2, anti-IRF3, and anti-phospho-IRF3 which were obtained from Cell Signaling Technology and used at a dilution of 1:1000.

The Caspase-Glo 1 assay (Promega) was used to measure the activity of caspase-1 in J774.1 macrophages either infected with M. tuberculosis at an MOI of 5 or treated with nigericin, an NLRP3 inflammasome inducer, at a final concentration of 20 μM. After 4–6 hours of infection or nigericin treatment, unprocessed cell samples were mixed at a 1:1 volume ratio with assay reagent in 96-well opaque plates, and the activity of caspase-1 in each sample was measured according to the manufacturer’s protocol. In brief, after 90 minutes of incubation at room temperature, luminescence produced by caspase cleavage of the Z-WEHD substrate was read on a standard plate reader. A caspase-1–specific inhibitor (Ac-YVAD-CHO) was added to parallel samples to confirm specific activity, and wells without cells were run to control for background signal. The values from both the Ac-YVAD-CHO and blank control were subtracted from the Z-WEHD substrate sample value, representing caspase-1 activity.
Immunoblots of cell lysates were probed with the following antibodies: anti-MDA5 (Cell Signaling Technology 3743), anti-LC3 (Cell Signaling Technology 12741), anti–caspase-1 (eBioscience 5B10), or anti–β-actin–HRP (Abcam 49900).

The Caspase-Glo 1 assay (Promega) was used to measure the activity of caspase-1 in J774.1 macrophages either infected with M. tuberculosis at an MOI of 5 or treated with nigericin, an NLRP3 inflammasome inducer, at a final concentration of 20 μM. After 4–6 hours of infection or nigericin treatment, unprocessed cell samples were mixed at a 1:1 volume ratio with assay reagent in 96-well opaque plates, and the activity of caspase-1 in each sample was measured according to the manufacturer’s protocol. In brief, after 90 minutes of incubation at room temperature, luminescence produced by caspase cleavage of the Z-WEHD substrate was read on a standard plate reader. A caspase-1–specific inhibitor (Ac-YVAD-CHO) was added to parallel samples to confirm specific activity, and wells without cells were run to control for background signal. The values from both the Ac-YVAD-CHO and blank control were subtracted from the Z-WEHD substrate sample value, representing caspase-1 activity.
Immunoblots of cell lysates were probed with the following antibodies: anti-MDA5 (Cell Signaling Technology 3743), anti-LC3 (Cell Signaling Technology 12741), anti–caspase-1 (eBioscience 5B10), or anti–β-actin–HRP (Abcam 49900).