<i>In vitro</i> protein-protein interaction assays.

<p>A) Pull-Down assay. The rEhCFIm25 immobilized on Ni²⁺-NTA column was incubated with NE. After washing, proteins were eluted, separated though 10% SDS-PAGE and stained with Coomassie Brilliant blue. Lane 1, Molecular weight markers; lane 2, proteins not retained on the column; lanes 3 and 4, washing fraction; lane 5, elution fraction. B) Far-Western assay. Purified rEhCFIm25 (lanes 1 to 4) and rEhPAP (lanes 5 to 8) were subjected to 10% SDS-PAGE and electrotransferred to a nitrocellulose membrane that was incubated with rEhPAP (lane 1) or rEhCFIm25 (lane 5). Proteins were immunodetected by specific antibody anti-EhPAP (lanes 1, 2 and 7) or anti-EhCFIm25 (lanes 3, 5 and 6) and revealed by the ECL Plus Western blotting system (Amersham). Anti-His antibody was used as control (lanes 4 and 8).