Mouse anti caspase 1 p20

Immunoblot analyses of cardiac tissue samples were carried out using a semi-quantitative western blotting analysis. The antibody used were: 1:1,000 rabbit anti-Ser^176/180-IKKα/β, 1:1,000 rabbit anti-total IKKα/β, 1:1,000 mouse anti-Ser^32/36-IκBα, 1:1,000 mouse anti-total IκBα, 1:1,000 rabbit anti-NF-κB, 1:1,000 rabbit anti-total BTK, 1:1,000 rabbit anti-Tyr¹²¹⁷ PLCγ, 1:1,000 rabbit anti-total PLCγ (from Cell Signaling), 1:1,000 rabbit anti-Tyr²²³-BTK, 1:5,000 rabbit anti NLRP3 inflammasome (from Abcam), 1:1,000 mouse anti-caspase 1 (p20) (from Adipogen). The apex of the heart was taken and homogenized in 1:10 of homogenization buffer at 4°C. Nuclear and cytosolic proteins were then extracted as previously described (21 (link)) and concentrations were quantified by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific Rockford, IL). Proteins were separated by 8% sodium dodecyl sulfate (SDS)-PAGE and transferred to polyvinyldenedifluoride membranes. Membranes were blocked in 10% milk solution with TBS-Tween and then incubated with the primary antibody overnight at 4°C. The next day the secondary antibody was added for 30 min at room temperature and visualized using the ECL detection system. Tubulin and histone 3 were used as loading control. The immunoreactive bands were analyzed by the Bio-Rad Image Lab Software™ 6.0.1 and results were normalized to the sham bands.

Immunoblot analyses of cardiac tissue samples were carried out using a semi-quantitative western blotting analysis. The antibody used were: 1:1,000 rabbit anti-Ser^176/180-IKKα/β, 1:1,000 rabbit anti-total IKKα/β, mouse anti-Ser^32/36-IκBα, mouse anti-total IκBα, rabbit anti-Tyr²²³-BTK, rabbit anti-total BTK, rabbit anti-Tyr¹²¹⁷ PLCγ, rabbit anti-total PLCγ (from Cell Signaling), 1:5,000 rabbit anti NLRP3 inflammasome (from Abcam), mouse anti-caspase 1 (p20) (from Adipogen). The apex of the heart was taken and homogenized. Proteins were then extracted as previously described (25 (link)) and concentrations were quantified by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific Rockford, IL). Proteins were separated by 8% sodium dodecyl sulfate (SDS)-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked in 10% milk solution with TBS-Tween and then incubated with the primary antibody overnight at 4°C. The next day the secondary antibody was added for 30 min at room temperature and visualized using the ECL detection system. Tubulin was used as loading control. The immunoreactive bands were analyzed by the Bio-Rad Image Lab Software™ 6.0.1 and results were normalized to the sham bands.

Proteins from lung tissues and THP-1 cells were extracted and analyzed by western blotting as described previously [12 (link)]. Total proteins were extracted with cell lysis buffer (Cell Signaling Technology, USA) according to the manufacturer’s instructions. Nuclear and cytosolic proteins were obtained with a commercial nuclear extraction kit (Thermo, USA) according to the manufacturer’s instructions. The primary antibodies used in this study included: mouse anti-NLRP3, mouse anti-caspase-1 (p20) (both from AdipoGen, San Diego, CA, USA), rabbit anti-RIP3, mouse anti-p-RIP3, rabbit anti-MLKL, rabbit anti-p-MLKL (all from Abcam, Cambridge, UK), mouse anti-RIP1 (R&D Systems, Minneapolis, MN, USA), rabbit anti-phospho-p44/42 MAPK kinase (p-ERK), total p44/42 MAPK kinase (t-ERK) and fibrillarin (all from Cell Signaling Technology, Beverly, MA, USA), and rabbit anti-GAPDH and anti-NF-κB p65 antibodies (both from Santa Cruz Biotechnology, Dallas, Texas, USA). HRP conjugated anti-mouse and anti-Rabbit IgG (both from Cell Signaling Technology, Beverly, MA, USA) were used as secondary antibodies. Signals were detected with enhanced chemiluminescence analysis kit (Cell Signaling Technology, Beverly, MA, USA).