FIG 2.

The 3C but not 2A protease of EV71 cleaves GSDMD. (A) 293T cells were transfected with plasmids encoding Flag-GSDMD-Myc along with GFP (lane 1) or increasing amounts of GFP-3C (lanes 2 to 5). At 24 h after transfection, the cells were then processed for Western blotting with antibodies, as indicated, using a Li-Cor Odyssey dual-color system (Li-Cor, Lincoln, NE). Antibodies recognizing Flag-GSDMD-Myc (Flag, N terminus of GSDMD, 800 nm, green; Myc, C terminus of GSDMD, 680 nm, red) were used. The merged images of the two channels are shown below (yellow). 3C or GFP was detected by using GFP antibody. β-Actin was included as a loading control. (B) 293T cells were transfected with plasmids encoding Flag-GSDMD-Myc along with a control plasmid or pcDNA3.1-IRES-2A plasmid. At 24 h after transfection, cells were lysed and analyzed by Western blotting. (C) The 3C protease interacts with GSDMD. 293T cells were transfected with plasmids encoding Flag-GSDMD (lanes 3 and 4), GFP (lanes 1 and 3), or GFP-3C (lanes 2 and 4). The total amount of DNA was kept constant with an empty vector. At 24 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody. Immunoprecipitates and aliquots of cell lysates were then analyzed by Western blotting. WCL, whole-cell lysates; IB, immunoblotting.