Immunoblot Analysis of Cardiac Proteins

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.