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. 2011 Nov 29;287(3):1892–1902. doi: 10.1074/jbc.M111.298687

FIGURE 3.

FIGURE 3.

Fn14 bound to MAP Ag85B. A, binding of fragments derived from the 40-kDa domain (including Fn12, Fn13, Fn14, and Fn15) to immobilized MAP Ag85B. GST-tagged Fn12, Fn13, Fn14, or Fn15 (0.1 μm) was added to microtiter plate wells coated with 0.5 μg of MAP Ag85B. Bound proteins were detected by ELISA. B, KD determination of Fn14 to MAP Ag85B by SPR analysis. The measured kon, koff, and KD values were 7.7 × 103 ± 1.8 m−1 s−1, 3.7 × 10−4 ± 0.6 s−1, and 48.1 ± 4.7 nm, respectively. Values represent the mean ± S.E. C, His-tagged MAP Ag85B was applied to the GST beads preimmobilized by recombinant Fn12 (lane 1), Fn13 (lane 2), Fn14 (lane 3), Fn15 (lane 4), or GST (lane 5; negative control). The pulldown complex was analyzed by immunoblot analysis using anti-His antibody. D, Fn14 interfered with the binding of MAP Ag85B to Fn. MAP Ag85B was preincubated with various concentrations (0.05, 0.1, 0.2, 0.5, and 1 μm) of Fn12, Fn13, Fn14, Fn15, or GST (negative control) prior to interaction with immobilized Fn. Bound proteins were detected by ELISA.