Figure 1.

Del-1 interferes with binding of α_v integrin to LAP. (A) Binding of α_vβ₆ integrin (100 nM) to immobilized BSA, Del-1 (100 nM), and LAP (100 nM), and binding of α_vβ₆ integrin to immobilized LAP in the presence of Del-1, as assessed in a solid-phase binding assay. Data are representative of three independent experiments, each with similar results, and are expressed as the mean ± SD (n = 4 per group). ***p < 0.001; Student's t-test. (B) Adhesion of α_vβ₆-overexpressing HEK293T cells or human small airway alveolar epithelial primary cells (HSAEpCs) onto increasing concentrations of immobilized LAP protein was assessed. Alternatively, adhesion of cells to immobilized inactive TGF-β derived from conditioned medium was assessed. Data are expressed as the mean ± SD (n = 3). *p < 0.05; Mann-Whitney U-test. (C) Adhesion of α_vβ₆ integrin-overexpressing HEK 293T cells or HSAEpC to immobilized LAP is impaired by Del-1 (1, 5, and 10 μg/mL), EGF₁₋₃ (5 μg/mL), or an α_vβ₆-blocking antibody (5 μg/mL). Data are expressed as the mean ± SD (n = 3). *p < 0.05; Mann-Whitney U-test. (D) Representative co-immunoprecipitation assay to validate the interaction between α_v integrin and Del-1 and the interaction between α_v integrin and LAP in the absence or presence of Del-1. HEK293T cells overexpressing α_vβ₆ integrin after lentiviral transduction were incubated for 6 h with Del-1 (20 nM) or LAP (20 nM) and then processed for immunoprecipitation and western blotting. Data are representative of three independent experiments, each with similar results.