Western Blot Analysis of AIM2, Caspase-1, and HPRT1

After 72 hpi, infected and mock-infected SK-N-SH and SK-N-SH/siAIM2 cells were rapidly washed with ice-cold PBS, scraped and centrifuged at 1000 × g for 10 min. Total proteins were extracted using Protein EX cell lysis buffer (GeneAll, Korea) supplemented with a phosphatase inhibitor, Pierce phosphotase inhibitor mini table (Thermo, USA) to prevent protein degradation and preserve their activation states. Protein concentration was determined by the Pierce BCA reagents (Thermo, USA), and 20 μg protein supernatant fractions were denatured and subjected to SDS-PAGE as described previously^{94 (link)}, with minor modifications. Briefly, the polyvinylidene fluoride membrane was incubated with AIM2 K-12 antibody (dilution 1:250; Santa Cruz, USA), Pierce EV-A71 Antibody (dilution 1:40000; Thermo, USA), caspase-1 antibody (dilution 1:100; Cell Signaling, USA), and HPRT1 antibody (dilution 1:500; Thermo, USA), respectively, overnight at 4 °C. Secondary goat anti-mouse IgG (31322) and goat anti-rabbit IgG (31342) antibodies conjugated with alkaline phosphatase (dilution 1:20000; Thermo, USA) were sequentially added. Blots were developed using 1-Step NBT/BCIP (Thermo, USA) at RT.