A Type III Effector NleF from EHEC Inhibits Epithelial Inflammatory Cell Death by Targeting Caspase-4

Figure 4

NleF inhibits caspase-4 activity by interrupting p19-p10 interaction. (a) GST-NleF or GST beads were mixed with lysates from E. coli cells expressing His-casp4-p19 or His-casp4-p10; the bound proteins were analyzed by immunoblotting with anti-His antibody. (b) Indicated purified proteins and GST-casp4-p19, or GST-casp1-p20 or GST-casp5-p20, were subjected to a competition assay. All bound proteins were analyzed by immunoblotting. (c) HT29 cells were transfected with Flag-casp4-p19 and Flag-casp4-p10 with Myc-NleF or Myc-NleF (Δ4aa). Supernatant aliquots were subjected to the cytotoxicity assay. Lysates of cells expressing Myc-NleF or Myc-NleF (Δ4aa) were analyzed by immunoblotting, and α-tubulin was used as an equal-loading control. (d) Purified His-casp4-p10, Flag-NleF (Δ4aa), and GST-casp4-p19 were subjected to a competition assay. All bound proteins were analyzed by immunoblotting. (e) 293T cells were cotransfected with Flag-caspase-1 and Myc-caspase-4 with HA-NleF or HA-NleF (Δ4aa); anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag antibodies. Graphs show the means ± SEM of triplicate wells; all data are representative of three independent experiments. . values were calculated using two-tailed Student’s -test.

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