Mapping and characterization of LigB1706–1716 binding sites on Fn. (A) A chart presenting Fn and truncated Fn used in this study. (B,C) Various concentration (0.625, 1.25, 2.5, 5, 10, 20, 40 μM) of biotin-LigB1706–1716 or biotin (negative control and data not shown) were added to 1 μg of (B) Fn, NTD, GBD, CBD, 40 kDa, or BSA (negative control) (C) 40 kDa, MBP-12–13F3, MBP-14F3, MBP-15F3, MBP (negative control), or BSA (negative control) coated microtiter plate wells. Bound proteins were measured by ELISA. (D) 1 μM of 15F3 in PBS in the presence of 0, 0.625, 1.25, 2.5, 5, 10, 20 and 40 μM of LigB1706–1716 was excited at 295 nm to measure Trp fluorescence. Inner plot: KD of 15F3-LigB1706–1716 determined by monitoring quenching fluorescence intensities at 350 nm. (E) Stop flow experiment of 15F3 binding to LigB1706–1716. The signal represents the total fluorescence emission above 320 nm by excited at 295 nm. Inner plot: The kinetic plot of kobs versus concentration of 1 μM 15F3 under different LigB1706–1716 concentrations (25, 50, 100, 200, 400 μM).