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. 2020 Mar 10;11:352. doi: 10.3389/fimmu.2020.00352

Figure 4.

Figure 4

Confirmation of inflammasome activation by viroporin via CRISPR-CAS 9. (A) HMEC-1 lysates expressing lentiCRISPRv2-GFP/Caspase/ASC were resolved by western blot. The primary antibody against ASC was used at a 1:1,000 dilution. HRP-anti rabbit was used as the secondary antibody (1:5,000). (B) ASC−/− HMEC-1 cells and WT HMEC-1 cells were primed with LPS 1 μg/mL for 6 h, followed by transfection with GFP, NS2A-GFP, and NS2B-GFP for 36 h. Positive control cells were primed with LPS 1 μg/mL for 6 h, followed by 5 mM ATP for 45 min. Lysates were analyzed using western blot. Caspase-1 primary antibody was used at a 1:1,000 dilution. HRP-anti rabbit was used as the secondary antibody (1:5,000). (C) HMEC-1 cells (WT or ASC−/−) were transfected with the expression plasmid encoding DENV-2 NS2A-GFP, NS2B-GFP or the pEGFPN1 parental vector for 36 h or treated with LPS (1 μg/mL) for 6 h followed by ATP (5 mM) for 45 min as a positive control, and the cell free supernatant was analyzed for IL-1β by ELISA. ***P < 0.001.