FIG. 1.
Expression of Tir in host cells results in its partial (phosphate-dependent) modification (A) HEK293 cells were transfected with a mammalian expression plasmid carrying a gene encoding TirHSV or infected with EPEC Δtir expressing TirHSV. 90°C-heat-soluble Triton X-100 fractions were isolated and incubated in the presence (+) or absence (−) of alkaline phosphatase. Samples were examined by Western analysis with probing with anti-HSV antibodies. (B) HEK293 cells expressing TirHSV were infected with EPEC or the Tir translocation-defective espA mutant. Infected cells were fractionated into saponin-released cytoplasmic (Cyto.), Triton X-100-soluble membrane (Mem.), or insoluble (Insol.) fractions and analyzed as described for panel A. (C) HEK293 cells were transfected with plasmids bearing genes encoding TirHSV or T7-TirHSV, and 90°C-heat-soluble Triton X-100 fractions were isolated. Samples were examining by Western analysis probing with anti-HSV, anti-Tir, or anti-T7 antibodies. T0, T′, and T" bands indicate the position of EPEC-delivered TirHSV intermediates, while H1-H4 indicate the position of mammalian-expressed Tir-related bands.