Expression of YopE in Y. pseudotuberculosis. (A) (Top) Western blot of steady-state expression level of YopE in Y. pseudotuberculosis grown at 37°C under nonsecreting conditions. YopE was detected with anti-YopE polyclonal antibodies. (Bottom) The number of cells analyzed was normalized, and as a loading control, DnaK was detected on the same blot with a monoclonal anti-DnaK antibody. YP126 is wild-type YopE expressed from its native locus on the pIB plasmid by Y. pseudotuberculosis 126 (YP126). WT, 3Ala, and 5Ala are wild-type YopE, YopE-3Ala, and YopE-5Ala, respectively, expressed from a low-copy-number plasmid by Y. pseudotuberculosis 126 ΔyopE. The last sample, ΔyopB, is wild-type YopE expressed from its native locus on the pIB plasmid by Y. pseudotuberculosis 126 ΔyopB. (B) Quantification of YopE expression levels under nonsecreting conditions, assessed by Western blotting. Mean values from four independent experiments carried out in triplicate are depicted. These values were normalized to the expression level of YP126 and are expressed as percentages of the YP126 value. Gray bars represent standard errors of the means.