Fig. 2

Characterization of the anti-cath-D F1 and E2 antibodies. a Binding of F1 and E2 to pro-cath-D secreted from MDA-MB-231 cells. Sandwich ELISA in which pro-cath-D from conditioned medium of MDA-MB-231 cells was added to wells pre-coated with anti-pro-cath-D M2E8 mouse monoclonal antibody in the presence of increasing concentrations of F1 (left panel) or E2 (right Panel). Binding of F1 and E2 to pro-cath-D was revealed with an anti-human Fc HRP-conjugated antibody. The EC₅₀ values are shown. b Binding of F1 and E2 to pro-cath-D secreted from MDA-MB-231 cells at acidic pH. Sandwich ELISA was performed as described in (a) but at different pH values (7.5–5.5). c Molecular docking of the scFv F1 and E2. Ribbon representation of the scFv F1 (magenta) and scFv E2 (green) interface with the contact surface of mature cath-D (upper panels). Docking model in which the space-filled view of protruding L1 CDR inserts into cath-D catalytic site (bottom panels). d Competitive ELISA. Sandwich ELISA was performed as described in (a) with 1 nM F1 or E2 and increasing concentrations of scFv F1, E2 or IR (negative scFv). e Immunoprecipitation of GST-cath-D isoforms with F1 and E2. GST-cath-D isoforms were immunoprecipitated with F1, E2, or E12, and detected by immunoblotting using the relevant antibodies (left panels). Mr, relative molecular mass (kDa). Schematic representation of the human 52-kDa pro-cath-D sequence (right panel). The 4-kDa cath-D pro-fragment, 14-kDa light, and 34-kDa heavy mature chains are indicated. The intermediate 48-kDa form (not shown) corresponds to the non-cleaved 14 + 34 kDa chains. The catalytic aspartate 33 and 231 (red) are shown