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: The levels of sEng in ( a ) plasma and in ( b ) serum were quantified using an ELISA kit assay in a cohort of 60 patients. This cohort comprised 20 non-pregnant women controls, 20 pregnant women controls, and 20 cases diagnosed with preeclampsia. c Plasma and ( d ) serum samples from preeclampsia patients diluted to ratios of 1:5, 1:10, or 1:20 and reduced with DDT, were subjected to analysis through SDS-PAGE and Western blot with Endoglin/CD105 polyclonal rabbit antibody (ProteinTech). rEng at 100 ng/mL was used as control. The data is presented as mean ± S.D. Statistical significance was determined at * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. NS indicates a non-specific band.

Article Snippet: Recombinant mouse Endoglin/CD105 Fc Chimera Protein, encompassing the extracellular region (Glu27-Gly581), was purchased from R&D Systems (1320-EN/CF). rEng (20 µg/mL) was diluted in Tris-buffered saline polyethylene glycol 1x buffer (TBS-PEG) and treated with human α-thrombin (Thr-H) (Cryopep, 9-HCT-0020-1) or human β-thrombin (Thr-C) (Cambridge ProteinWorks, #10108).

Techniques: Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Control

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: Recombinant mouse Endoglin/CD105 Fc Chimera Protein, encompassing the extracellular region (Glu27-Gly581), was purchased from R&D Systems (1320-EN/CF). rEng (20 µg/mL) was diluted in Tris-buffered saline polyethylene glycol 1x buffer (TBS-PEG) and treated with human α-thrombin (Thr-H) (Cryopep, 9-HCT-0020-1) or human β-thrombin (Thr-C) (Cambridge ProteinWorks, #10108).

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 Immunofluorescence staining of Endoglin was conducted using the Endoglin/CD105 P4A4-Alexa 488 antibody (green) and vectashield mounting medium with DAPI (blue) after treating ECFC with increasing concentrations of Thr-H (0.01, 0.1 and 1 µM). Observations were carried out using confocal microscopy at 20x magnification. Phase photos at 10x magnification were taken for each condition to confirm the presence of the cell monolayer. b Endoglin fluorescence intensity following ECFC treatment with increasing concentrations of Thr-H (0.01, 0.1 and 1 µM) was quantified using ImageJ software ( n = 3). c Plots showing the quantification of Eng fluorescence intensity in ECFC following treatment with 1 µM of Thr-H, alongside the number of nuclei relative to the surface. d Immunofluorescence staining of Eng using the Endoglin/CD105 P4A4-Alexa 488 antibody in different cell types, including HUVECs and MSCs, mounted with Vectashield containing DAPI. HUVECs were additionally stained with VE-Cadherin (VE-CAD) using a Texas Red-conjugated antibody, and MSCs were stained with Phalloidin-Alexa 555 to assess cell integrity after treatment with Thr. e Plots showing the quantification of Eng fluorescence intensity in HUVEC and MSC following treatment with 1 µM of Thr-H, alongside the number of nuclei relative to the surface. f ELISA kit assay results for sEng in supernatants of ECFC, HUVEC and MSC treated or not with 1 µM of Thr-H. The data is presented as mean ± S.D. for a minimum of n = 4. Statistical significance was considered at * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Recombinant mouse Endoglin/CD105 Fc Chimera Protein, encompassing the extracellular region (Glu27-Gly581), was purchased from R&D Systems (1320-EN/CF). rEng (20 µg/mL) was diluted in Tris-buffered saline polyethylene glycol 1x buffer (TBS-PEG) and treated with human α-thrombin (Thr-H) (Cryopep, 9-HCT-0020-1) or human β-thrombin (Thr-C) (Cambridge ProteinWorks, #10108).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Fluorescence, Software, Enzyme-linked Immunosorbent Assay