Affinity interaction between EscA with CesA2, between EscA and EscL, and between EscA and EscN as demonstrated by co-elution from a Ni²⁺-NTA column.

Experiments were carried out in a manner similar to that in Figure 3 with a slight modification. In essence, a His_×6 tag was placed at the C-terminal end of CesA2 (A), EscL (B), YgfZ (C), EscA (D), and EscN (E). Bacterial lysate containing the His_×6-tagged protein was individually applied to the Ni²⁺-NTA column. Then bacterial lysate containing tag-free EscA (A-C and E) or CesAB (D) was applied to the column. After washing and eluting, fractions were analyzed by Western blotting with specific antibodies except that all His_×6-tagged proteins were all detected by anti-His_×6. I: input; F: flow through.