Anti caspase 3 and 9

After protein concentrations were determined using bicinchoninic acid, equal amounts of protein lysates were separated by SDS-PAGE and electrotransferred onto nitrocellulose membranes followed by blockade with 3% BSA. Subsequently, membranes were incubated with anti-caspase-3 and -9 (1:500, #9662 and #9506; Cell Signaling Technology, Beverly, MA, USA), anti-Cytochrome C and anti-p53 (1:400, #4280 and #32532; Cell Signaling Technology, Beverly, MA, USA), anti-caspase-11 (1:1000, sc-28230; Santa Cruz Biotechnology, CA, USA), anti-caspase-1 (1:1,000, sc-1218, Santa Cruz Biotechnology, CA, USA), anti-NLRP3 (1:1000, ab214185, Abcam, Cambridge, MA, USA), or anti-β-actin (1:1000, #4970, Cell Signaling Technology, Beverly, MA, USA), anti-GAPDH (1:5000, sc-20358, Santa Cruz Biotechnology, CA, USA), and anti-VDAC (#4866, 1:5,000, Cell Signaling Technology, Beverly, MA, USA) at 4°C overnight. After membranes were washed, bound antibodies on membranes were immersed in horseradish peroxidease-conjugated secondary antibody (1:1,000) at room temperature for 1 h and visualized using enhanced chemiluminescence. Relative level of each protein to control protein was quantified using Quantity One software (Bio-Rad Laboratories, Hercules, CA).