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).
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).