Co-elution of EspA and EscA.

<p>(A) Schematic illustration of the expression constructs. (B) Affinity co-elution analysis of Ni<sup>2+</sup>-NTA column-retained proteins. In panels I and II, bacterial lysates from plasmid-transformed <i>E. coli</i> JM109 were loaded onto the column. After washing with buffer containing 50 mM imidazole, the retained proteins were eluted with the same buffer containing 250 mM imidazole. The presence of EspA from the individual fractions of the panels was detected by Western blotting using rabbit anti-EspA antibodies. (C) Analysis of EspB in elution fractions of the Ni<sup>2+</sup>-NTA column in the presence of His<sub>×6</sub>-tagged EscA binding. Experiments were carried out in a manner similar to that in (B) except that the bacteria were transformed with p<i>T5</i>-EspB_His-EscA and EspB was detected with rabbit anti-EspB antibodies.</p>