Journal: Communications Biology

Article Title: Thrombin cleaves membrane-bound endoglin potentially contributing to the heterogeneity of circulating endoglin in preeclampsia

doi: 10.1038/s42003-025-07751-3

Figure Lengend Snippet: rEng at a concentration of 20 µg/mL was treated at 37 °C a with increasing concentrations of Thr-H : 0, 0.01, 0.05, 0.1, 0.5, or 1 µM for 1 h. Following treatment, the samples were resolved using SDS-PAGE and subsequently analyzed by Western blotting with Endoglin/CD105 polyclonal rabbit antibody (ProteinTech). b The percentage of all cleaved bands obtained in a by the action of Thr-H on Eng was quantified relatively to the total rEng (70 kDa). c Percentage of the 70 kDa band reduction, specific to the total rEng band from a . d The same procedure described in a was repeated (Eng 20 µg/mL, treatment at 37 °C), but using a fixed concentration of 1 µM of Thr-H for varying durations: 0, 1, 5, 15, 30 or 60 min. e Graph of percentage of all cleaved bands resulting from the action of Thr-H on rEng at various time intervals. f Percentage of the 70 kDa band reduction, specific to the total rEng band from d . left. In a and d the band above 40 kDa (of approximately 50 kDa) was not reproducibly observed in the repeated experiments and was not further studied. The data is presented as mean ± S.D. from three conducted experiments. All images analysis were performed using Image Studio™ Lite software. The dashed line in panel d indicates a cut made in the gel where a blank column had been.

Article Snippet: Fig. 3 Dose and time dependent cleavage of human rEng by thrombin. rEng at a concentration of 20 µg/mL was treated at 37 °C a with increasing concentrations of Thr-H : 0, 0.01, 0.05, 0.1, 0.5, or 1 µM for 1 h. Following treatment, the samples were resolved using SDS-PAGE and subsequently analyzed by Western blotting with Endoglin/CD105 polyclonal rabbit antibody (ProteinTech). b The percentage of all cleaved bands obtained in a by the action of Thr-H on Eng was quantified relatively to the total rEng (70 kDa). c Percentage of the 70 kDa band reduction, specific to the total rEng band from a . d The same procedure described in a was repeated (Eng 20 µg/mL, treatment at 37 °C), but using a fixed concentration of 1 µM of Thr-H for varying durations: 0, 1, 5, 15, 30 or 60 min. e Graph of percentage of all cleaved bands resulting from the action of Thr-H on rEng at various time intervals. f Percentage of the 70 kDa band reduction, specific to the total rEng band from d . left.

Techniques: Concentration Assay, SDS Page, Western Blot, Software

Journal: Communications Biology

Article Title: Thrombin cleaves membrane-bound endoglin potentially contributing to the heterogeneity of circulating endoglin in preeclampsia

doi: 10.1038/s42003-025-07751-3

Figure Lengend Snippet: 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.

Article Snippet: Fig. 3 Dose and time dependent cleavage of human rEng by thrombin. rEng at a concentration of 20 µg/mL was treated at 37 °C a with increasing concentrations of Thr-H : 0, 0.01, 0.05, 0.1, 0.5, or 1 µM for 1 h. Following treatment, the samples were resolved using SDS-PAGE and subsequently analyzed by Western blotting with Endoglin/CD105 polyclonal rabbit antibody (ProteinTech). b The percentage of all cleaved bands obtained in a by the action of Thr-H on Eng was quantified relatively to the total rEng (70 kDa). c Percentage of the 70 kDa band reduction, specific to the total rEng band from a . d The same procedure described in a was repeated (Eng 20 µg/mL, treatment at 37 °C), but using a fixed concentration of 1 µM of Thr-H for varying durations: 0, 1, 5, 15, 30 or 60 min. e Graph of percentage of all cleaved bands resulting from the action of Thr-H on rEng at various time intervals. f Percentage of the 70 kDa band reduction, specific to the total rEng band from d . left.

Techniques: Concentration Assay, SDS Page, Staining, Sequencing, Molecular Weight, Software, Western Blot

Journal:

Article Title: Lipopolysaccharide is a Frequent and Significant Contaminant in Microglia-Activating Factors

doi: 10.1002/glia.20585

Figure Lengend Snippet: Quantification of LPS (Endotoxin) Levels in Various Commercial- and Pharmaceutical-Grade Preparations and Reagents

Article Snippet: They also thank ZymoGenetics, Seattle, WA, USA and GTC Biotherapeutics, Framingham, MA, USA for the generous gifts of pharmaceutical-grade recombinant human α-thrombin and antithrombin, respectively.

Techniques: Activity Assay, Functional Assay, Recombinant, Clinical Proteomics