FIG 4.
Effect of the Y219A and E253A mutations on NleB1 translocation from EPEC and caspase-8 activation during infection. (A) Translocation of NleB1 derivatives NleB1PILN(63–66)AAAA, NleB1PDG(236–238)AAA, NleB1Y219A, and NleB1E253A fused to TEM-1 from EPEC ΔnleB1. Results are the means ± SEMs from three independent experiments carried out in triplicate. Significant differences were determined by one-way analysis of variance with Tukey's multiple-comparison test. ***, P < 0.0001. (B) Immunoblot showing cleaved caspase-8 in HeLa cells infected with EPEC derivatives that expressed the different NleB1 mutants [NleB1DXD(221–223)AxA, NleB1Y219A, NleB1E253A] from the pTrc99A vector backbone and that were stimulated with FasL. Cells were harvested for immunoblotting, and cleaved caspase-8 was detected with anti-cleaved caspase-8 antibodies. Antibodies to β-actin were used as a loading control. The immunoblots are representative of those from at least three independent experiments.