Aim2 k 12 antibody

EV-A71/13903-infected SK-N-SH and AIM2-knockdown SK-N-SH cells (SK-N-SH/siAIM2) cells at MOI of 1, and mock-infected cells were triple-stained for EV-A71 antigens, AIM2 and 7 amino-actinomycin (7-AAD). Previously shown to be useful as a biomarker for pyroptosis, 7-AAD is able to enter nuclear membranes to stain DNA following the loss of membrane integrity^{41 (link), 65 (link), 91 (link)}. Briefly, infected and mock-infected cells were collected in 1.5 ml microcentrifuge tubes and incubated for 5 minutes with 7-AAD^{87 (link)–89 (link)}. The cell pellets were then fixed with Fluorofix buffer and stained for viral antigen and AIM2 protein were using mouse monoclonal Enterovirus blend 3321 antibody and rabbit polyclonal AIM2 K-12 antibody (dilution 1:500; Santa Cruz, USA), followed by incubation with by goat-anti mouse IgG conjugated with Alexa-fluor 488 and goat anti-rabbit IgG conjugated with Alexa-fluor 546 (Molecular Probes, USA) for 30 min at RT in the dark^{92 (link)}. Flow cytometry analysis to assess localization of viral antigens and AIM2 or 7-AAD was performed as before.

The CNS tissues of 3 confirmed EV-A71 encephalomyelitis cases from a previous autopsy series^{22 (link), 95 (link)} were investigated by IHC for AIM2 protein expression. IHC was performed by a standard ENVISION technique as described previously^{96 (link)}. Briefly, deparaffinized and rehydrated tissue sections were endogenous peroxidase blocked before antigen retrieval at 30 min in Tris-EDTA buffer (pH 9) with 0.05% Tween-20. Tissue sections were then incubated with 10% normal goat serum before incubation overnight at 4 °C with mouse monoclonal Enterovirus blend 3321 antibody (dilution 1:50; Merck, Germany) or rabbit polyclonal AIM2 K-12 antibody (dilution 1:500; Santa cruz, USA). Secondary goat-anti mouse IgG HRP-conjugated (Dako, Denmark) or goat-anti rabbit IgG HRP conjugated (Dako, Denmark) respectively, was applied, followed by DAB (Dako, Denmark) chromogen. The slides were counterstained with hematoxylin (Dako, Denmark) and mounted with DPX mounting medium (Sigma, USA). Negative controls for IHC were normal human brain tissues. Positive controls for AIM2 staining were normal human small intestine and for EV-A71 antigens, EV-A71/13903 infected SK-N-SH cells. Duplicate assays on test tissues were also done by replacing the primary antibodies with isotype control antibodies or normal rabbit immunoglobulin fractions (l) (Dako, Denmark).

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.