Fig. 4: Sequencing of thrombin-cleaved endoglin bands.

a rEng at a concentration of 20 µg/mL was treated at 37 °C for 1 h with 1 µM of Thr-H. The samples were then resolved by SDS-PAGE and subjected to Coomassie Brilliant Blue staining to identify protein spots for subsequent sequencing. Bands at molecular weights of 60, 40, 20, 10, and 8 kDa were excised, with a minimum of 30 bands at each molecular weight selected for further analysis. Plots showing b the decrease in the percentage of the 70 kDa band, specific to the total rEng band and c the percentage of all cleaved bands obtained by the action of Thr-H on Eng, quantified relatively to the total rEng (70 kDa). The optical densities of each Coomassie blue-stained protein band were measured using Image Studio™ Lite software. The data is presented as mean ± S.D. from a minimum of 12 conducted experiments. Statistical significance was considered at *P < 0.05. d Table showing the N-terminal and C-terminal parts of each sequenced band, along with the cleavage position and the cleaved amino acids. The C-terminal information was not determined (ND) for the 8, 40 and 60 kDa bands, and the cleavage position and cleaved amino acids for the last two bands were also unidentified. However, based on the docking data and the Western blot analysis with the monoclonal antibody MAB1097 (R&D), that specifically labels the endoglin orphan region, for we can postulate that the 40 KDa band corresponds to the fragment 329–330. e Schematic diagram illustrating the N-terminal cleavage positions and sequences of each band, including known (solid line) and hypothetical (dashed line) C-terminal cleavages. Because the contribution of glycosylation to the molecular weight of each band is unknown, the correlation between molecular weight and primary structure is not to scale. f Schematic diagram of mEng, indicating cleavage sites. mEng is composed of an orphan region followed by a juxtamembrane zona pellucida (ZP) module and by transmembrane and short cytoplasmic domains. JM: Juxtamembrane; TM: Transmembrane; CP: Cytoplasmic.