Fig. 1.

Deletion of exsE does not negatively affect synthesis or secretion of Vp1656. (a) The indicated strains were used to infect HeLa cells cultured in DMEM supplemented with 10 % (v/v) FBS at an m.o.i. of 100 for 4 h followed by centrifugation to separate cell pellet and supernatant fractions. Cell-associated and secreted proteins obtained from whole-cell lysate and trichloroacetic acid-precipitated cell-free supernatant, respectively, were separated using 4–20 % SDS-PAGE, transferred to PVDF and probed using anti-Vp1656 and anti-DnaK antibodies. DnaK was used as a loading control. This experiment was replicated three times and a representative blot is shown here. (b) RT-PCR was used to detect mRNA transcripts for a subset of T3SS1 genes after infection. RT+ reactions included reverse transcriptase; RT− reactions contained no reverse transcriptase. The latter indicated no evidence for contaminating DNA in the reaction.