Expression and stability of EspA are decreased in the absence of <i>escA.</i>

<p>(A) Detection of EspA and EspB that were ectopically expressed and detected by Western blotting using anti-His<sub>×6</sub>. EHEC (WT) and mutant Δ<i>escA</i> were transformed with the plasmids indicated and the bacteria were cultured for 6 h at 37°C in M9-5% CO<sub>2</sub>. Proteins from the total bacterial lysates were analyzed by Western blotting using rabbit anti-His<sub>×6</sub> antibodies. The dotted box indicates the expected banding area of EspA. Multiple bands of EspB detected could be the results of degradation presumably arising from overexpression. (B) Stability of EspA in the bacteria, as reflected by sample analyses over 16 h after <i>de novo</i> protein syntheses of bacteria were blocked by addition of chloramphenicol. EspA was analyzed in a way similar to that of (A). (C) The amount of EspA over the time period in (B) was converted into a stability curve by quantifying the band intensities using a densitometer. The EspA intensity at time zero is referred to as 100% while simultaneously detected OmpC was used for loading calibration. (D) Expression of CesA2 and EscL not affected by the lack of EscA in bacteria. Bacteria were transformed with pQE-CesA2 and pQE-EscL, respectively. Bacterial cultures and detection of proteins were carried out similar to that in (A).</p>