Figure 5

LTBP-1 binds to immobilized Notch3 in a solid-phase binding assay. a The interaction of full-length LTBP-1 derived from conditioned cell supernatants with an immobilized Notch3 fragment (N3EGF1-11-Fc) is increased 4.9-fold when compared to a control ligand (IgG-Fc). Results are expressed as mean + SEM of seven independent experiments. Bound LTBP-1 was re-evaluated by immunoblotting and predominantly detected in oligomeric form. b Schematic representation of the used LTBP-1 constructs and their domain organization including cysteine-rich repeats (orange ovals), a hybrid domain (grey ovals), EGF-like repeats (blue boxes) and V5-His or HA tags (black circles). The fibronectin, TGF-β, and fibrillin-1 binding regions are indicated. Note that LTBP-ΔC-HA was used in assays with cell supernatants and LTBP-ΔC-V5 in assays requiring purified LTBP-1. c LTBP-1 binding to Notch3 is mediated by its N-terminus. While the N-terminal deletion variant LTBP-1ΔN does not bind significantly, the C-terminal deletion variant LTBP-1ΔC, either from conditioned supernatant or in purified form, shows significant interaction. Results are expressed as mean + SEM of five (LTBP-1ΔC_V5) and four (LTBP-1ΔN_HA) independent experiments. n.s.: not significant, ***p < 0.001; **p < 0.01; Mann–Whitney Test.