FIG. 5.

Column binding assay showing that CesF specifically binds EspF but not other secreted EPEC proteins. (A) Proteins secreted from EPEC were passed over an Ni²⁺-NTA column containing prebound His₆CesF. EspF was retained on the column and was eluted along with CesF (lanes 6 to 9). In contrast, other secreted proteins (Tir, EspA, EspB, EspC) were not retained and flowed through the column (lane 2), although a small amount of Tir was detected, consistent with a nonspecific interaction with the column (lane 6). (B) EspF is not retained on Ni²⁺-NTA resin in the absence of CesF. All proteins were detected by Western blotting with specific antiserum as described in the text.