FIGURE 2.

Identification and characterization of the FMOD-LOX interaction. A, proximity ligation assay on HFL-1 cells expressing FMOD (left panel) or non-collagen-binding His-tagged FMOD fragment as a negative control (right panel). Anti-His tag and anti-LOX antibodies were used to detect the interaction (red fluorescence). Nuclear staining with DAPI is shown in blue. B, gel image of FMOD variants used in the solid-phase assays in C–F. The proteins were expressed in 293T cells and purified on Ni-NTA-Sepharose. The amino acid (aa) range of each construct is listed. C, solid-phase assays showing interactions between FMOD variants and LOX. LOX was coated on a 96-well plate that was blocked with BSA and incubated with 170 nm FMOD variants, whose binding was detected using anti-His tag antibody. D, as in C, but here FMOD variants were coated on the plate followed by incubation with 150 nm LOX and detection with anti-LOX antibody. The experiments in C and D were repeated twice, giving similar results. E, as in D, but here serial dilutions of LOX were used to determine the apparent K_D. Similar results were obtained in three experiments. F, solid-phase assay to assess binding of LOX to coated FMOD (expressed in 293 cells), bactFMOD (expressed in E. coli), and FMOD90. The assay was performed as in D. G, solid-phase assay to compare binding of serially diluted FMOD and LOX to coated collagen in a 96-well plate. Binding was detected using anti-His tag or anti-LOX antibodies. All error bars represent mean ± S.D. (n = 3 technical replicates). The experiments in F and G gave similar results on three occasions.