Characterizing the effects of the <i>escA</i> deletion on the detection of representative <i>LEE</i> proteins.

<p>(A) Schematic diagram of the gene organization around <i>LEE3</i> and an illustration of how the deletion of <i>escA</i> in mutant strain (Δ<i>escA</i>) was created. Bent arrow indicates the transcription direction of the <i>LEE3</i> operon. (B) Detection of representative LEE proteins (Tir, EspB, and EspA) by Western blotting using specific antibodies. Bacteria transformed with pQE-EscA or control vector (pQE-60) were grown in M9 in the presence of 5% CO₂. (C) Secretion of effectors Map and EspF also affected by the <i>escA</i> delection. Bacteria were separately transformed with pQE-Map and pQE-EspF, and the His_×6-tagged proteins in the bacterial lysates and the culture supernatants were detected similar to that in (B) except for the excusive usage of anti-His_×6 antibodies. Bacterial lysate and concentrated supernatant were prepared separately and analyzed accordingly. OmpC was used as the protein loading control for the bacterial lysate samples. Note: dot denotes a detected non-specific signal above the band of Tir whereas asterisk indicates a band presumably arising from His_×6-tagged EspF that was abundantly expressed.