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. 2011 Jul 1;286(34):29922–29931. doi: 10.1074/jbc.M111.233858

FIGURE 2.

FIGURE 2.

Far Western probing to examine binding of ME0052-Bio to putative target proteins. A, putative target proteins were cloned into pET151 and expressed in E. coli BL21 (λDE3) cells before lysis, separation, and transfer to a nitrocellulose membrane. ME0052-Bio was used as a probe, and interactions were detected using HRP-conjugated streptavidin. Proteins probed were (or encoded by) Tpx (1), FolX (2), z2714 (3), z3974 (4), WrbA (5), SurA (6), and StcE (7). Lane 8 contains bacterial lysate with no overexpressed protein as a negative control. The same protocol was used to subsequently probe proteins from E. coli O157 (EC), Y. pseudotuberculosis (YP), S. typhimurium (ST), P. aeruginosa (PA), and S. flexneri (SF) or empty vector control (−ve). Panels show overexpression of target proteins: WrbA (B), Tpx (C), FolX and FolB (D). Mutation of Tpx Cys-61 (C61S) affected binding of ME0052-Bio (E). The addition of 200 μm unlabeled ME0052 (F–H) strongly affected the binding of the biotinylated probe.