Analysis of heterotypic interaction of MUC5B dimerization and multimerization domains.
A, a pH scan of 50 nm NT5B in different pH buffers in the presence of 5 mm CaCl2, flowed over CT5B immobilized to a chip and analyzed using SPR. There was some background binding at pH 7.4, but strong binding occurred between NT5B and CT5B at pH ∼ 6.2. This experiment was performed once with triplicate samples. B, single cycle kinetic analysis with increasing concentrations of NT5B in 5 mm CaCl2 (0, 5, 10, 15, 20, and 40 nm) at pH 6 (black line) or pH 7.4 (dashed line). C and D, serially diluted recombinant CT5B protein was mixed with an equal volume of fluorescently labeled NT5B recombinant protein, and interactions were measured by fluorescence using MST. Samples were prepared in HBS buffer containing 0.05% Tween 20 and either 5 mm CaCl2 (circles) or 5 mm EGTA (triangles) at pH 7.4 (C) or 6 (D). Error bars represent the mean ± S.E. Experiments in B–D were repeated at least twice.