Anti cleaved il 1β

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.

Cell Signaling Technology, Inc. (Danvers, MA, USA) supplied the following antibodies: anti-nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) p65 subunit antibody (cat. 8242; dil. 1:1,000); anti-NACHT, LRR and PYD domains-containing protein 3 (NLRP3) antibody (cat. 13158; dil. for WB 1:500 and for IF 1:100); anti-caspase 1 (CASP1) antibody (cat. 2225; dil. 1:1,000), anti-cleaved-IL-1β (cat. 83186; dil. 1:100); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (cat. 5174; dil. 1:5,000); anti-histone deacetylase 1 (HDAC1) antibody (cat. 8242; dil. 1:1,000), and peroxidase-conjugated anti-rabbit secondary antibody (cat. 7074; dil. 1:10,000). Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) provided the anti-apoptosis-associated speck-like protein containing CARD (ASC) (cat. sc-271054; dil. for WB 1:800 and for IF 1:200) antibody. Both AlexaFluorTM 488 goat anti-rabbit IgG (H + L) (cat. A-11008; dil. 1:1,000) and AlexaFluorTM 568 goat anti-mouse IgG (H + L) (cat. A-11004; dil. 1:1,000) antibodies were provided by Thermo Fisher Scientific (Waltham, MA, USA). The peroxidase-conjugated anti-mouse secondary antibody (cat. 170–6515; dil. 1:10,000) was supplied by Bio-Rad Laboratories (Hercules, CA, USA).

Total proteins were obtained by using radio immunoprecipitation assay (RIPA) buffer (Thermo Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail (protease inhibitor cocktail 1 tablet; Roche Diagnostics, Mannheim, Germany) and centrifuged at 13,000 rpm for 10 min. The protein was measured with BCA Protein Assay Kit by using microplate reader at 562 nm. Equal amounts of protein were separated on 10% SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% bovine serum albumin, protein was probed with appropriate antibodies.
The primary antibodies used were as follows: anti-TRAP, anti-cathepsin K, anti-c-Fos, anti-NFATc1, anti-TRAF6, and anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-carbonic anhydrase II, anti-IκBα, and anti-phospho-IκBα from Abcam (Cambridge, MA, USA); and anti-IL-1β and anti-cleaved IL-1β from Cell Signaling Technology (Danvers, MA, USA). The SuperSignal® West Pico chemiluminescent kit (Thermo Scientific, Rockford, IL, USA) was used to detect the target protein. Images were obtained using a ChemiDoc TM XRS system (Bio-Rad Laboratories, Hercules, CA, USA).