FIG. 4.

Interaction of M. pneumoniae EF-Tu FL and its truncations with different Fn domains. (A) Schematic representation of different domains of Fn. The figure was adapted from Virkola et al. (53). Fn domains used for the ligand blot analyses are indicated in cross-hatched boxes. (B) Interaction of EF-Tu FL, ED3, and ED4 with domains of Fn. Equal amounts of available Fn fragments were separated individually on 4 to 12% Nu-PAGE gels and transferred onto nitrocellulose membranes, which were incubated with 10 μg/ml recombinant EF-Tu proteins in 3% Blotto in 1× TBS for 2 h at RT (see Materials and Methods). Membranes were probed with mouse anti-His MAb (1:5,000) and subsequently with goat anti-mouse AP (1:2,000) reagent. Based on the mean intensities of His tag antibody recognition of recombinant EF-Tu proteins, interactions were classified as demonstrating weak (+), intermediate (++), strong (+++) or no (−) binding.