FIGURE 4.

Deciphering the mode of action of pro-domain displacement, investigated by Biacore sandwich complex formation assays. To further dissect the interaction of BMP9 variants with ALK1, sandwich complex formation SPR assays were designed. The illustration in A visualizes the assay setup. ALK1-Fc was captured via Fc part on a CM5-Chip. BMP9 variants and detection antibodies α-mature BMP9-mAb MAB3209 or α-pro-domain-Ab AF3879 were injected consecutively using a dual inject. Sensorgrams for complex formations are shown for mature BMP9 (B), BMP9·pro-domain (C), precursor BMP9 (D), and pro-domain (E) as the first analyte injected. After complex formation of the receptor with BMP9 variants on the chip surface, the pro-domain was only detected for the precursor BMP9 by pro-domain-specific antibody AF3879 in a subsequent injection (D).