Journal: The Journal of Biological Chemistry

Article Title: Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains *

doi: 10.1074/jbc.M115.680009

Figure Lengend Snippet: Release of mature growth factor from BMP9·prodomain complex. Release of mature BMP9 domain after binding of an anti-pro-domain antibody was confirmed by application of BMP9·pro-domain to the pro-domain ELISA using anti-pro-domain antibodies AF3879 as capture and BAF3879 as detection (A). B, supernatant from A and recombinant mature BMP9 standard were applied to the mature BMP9 ELISA using anti-mature BMP9 antibodies MAB3209 as a capture and BAF3209 as detection. The mature BMP9 ELISA detected both mature BMP9 standard and released mature BMP9 from pro-domain ELISA supernatant.

Article Snippet: Antibodies used included anti-mature BMP9 antibodies MAB3209 and its biotinylated conjugate BAF3209 and anti-pro-domain antibodies AF3879 and its biotinylated conjugate BAF3879 and were purchased from R&D Systems.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: EBioMedicine

Article Title: Reduced circulating BMP10 and BMP9 and elevated endoglin are associated with disease severity, decompensation and pulmonary vascular syndromes in patients with cirrhosis

doi: 10.1016/j.ebiom.2020.102794

Figure Lengend Snippet: Reduced plasma BMP9 and pBMP10 levels and increased sEng levels in cirrhosis are associated with decompensation. (A,D,G) Plasma samples from controls or patients with either pre-cirrhotic liver fibrosis or with cirrhosis were assayed by ELISA for (A) BMP9, (D) pBMP10 or (G) sEng. Error bars show median and interquartile range. (B,E,H) Spearman correlation of: (B) BMP9 ( n = 69, P< 0.0001), (E) pBMP10 ( n = 64, P< 0.0001) or (H) sEng ( n = 58, P< 0.0001) levels with MELD-Na score of cirrhosis severity. (C,F,I) Patients were stratified for compensated and decompensated liver disease and plasma levels of (C) BMP9, (F) pBMP10 and (I) sEng compared between groups. Kruskal Wallis test: * P< 0.05, *** P< 0.001, **** P< 0.0001, Mann Whitney Test # P< 0.05.

Article Snippet: Primary antibodies against BMP9 (AP2064A, 1:50 Abcepta, San Diego, CA), BMP10 (LS-C293026, 1:100, LSBio, Seattle, WA) and hepatocyte antibody (OCH1E5, 1:20, Thermo Fisher, Waltham, MA) were then added to human liver slides in PBS/Tween, 3% goat serum at 250 µl per slide.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: EBioMedicine

Article Title: Reduced circulating BMP10 and BMP9 and elevated endoglin are associated with disease severity, decompensation and pulmonary vascular syndromes in patients with cirrhosis

doi: 10.1016/j.ebiom.2020.102794

Figure Lengend Snippet: Circulating BMP9 and pBMP10 levels are reduced in PoPH and HPS, whereas sEng levels are elevated in both syndromes. Plasma samples ELISA data were for (A) BMP9, (B) pBMP10 or (C) sEng in cirrhotic patients were allocated according to the presence or absence of HPS, with controls shown for reference. Plasma samples from a separate cohort of controls and patients with confirmed PoPH were assayed by ELISA for (D) BMP9, (E) pBMP10 or (F) sEng. (G–I) The datasets for the control group samples and PoPH patients form both cohorts were combined and analysed to assess the levels of (G) BMP9, (H) pBMP10 and (I) sEng in the overall PoPH cohort. The data for the HPS patients are included in these graphs for reference. Error bars show median and interquartile range. Kruskal Wallis test: * P< 0.05, ** P< 0.01, *** P< 0.001, **** P< 0.0001, Mann Whitney Test: # P< 0.05.

Article Snippet: Primary antibodies against BMP9 (AP2064A, 1:50 Abcepta, San Diego, CA), BMP10 (LS-C293026, 1:100, LSBio, Seattle, WA) and hepatocyte antibody (OCH1E5, 1:20, Thermo Fisher, Waltham, MA) were then added to human liver slides in PBS/Tween, 3% goat serum at 250 µl per slide.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: EBioMedicine

Article Title: Reduced circulating BMP10 and BMP9 and elevated endoglin are associated with disease severity, decompensation and pulmonary vascular syndromes in patients with cirrhosis

doi: 10.1016/j.ebiom.2020.102794

Figure Lengend Snippet: BMP activity is low in liver disease but can be reconstituted with exogenous BMP9. (A,B) HAECs were serum-depleted overnight followed by addition of 1 ng/ml BMP9, BMP10, or 1% plasma from controls ( n = 6) or liver disease patients with undetectable (320; n = 5) levels of BMP9. After 1 h, cells were lysed, RNA extracted and cDNA analysed for the expression of (A) ID1 or (B) ID2 . (C and D) Plasmas (1% final concentration) from controls ( n = 4) or liver disease patients ( n = 4) were added alone, or after spiking with ProBMP9, to serum depleted HAECs for 1 h. After 1 h, cells were lysed, RNA extracted, and cDNA analysed for the expression of (C) ID1 or (D) ID2 . (E) The same spiked plasma samples ( n = 4 control and 4 liver disease) as in (C) and (D) were assayed using the BMP9 ELISA. * P< 0.05, ** P <0.01, *** P< 0.001 Kruskal Wallis test, # P< 0.05 Mann–Whitney test compared to control plasma.

Article Snippet: Primary antibodies against BMP9 (AP2064A, 1:50 Abcepta, San Diego, CA), BMP10 (LS-C293026, 1:100, LSBio, Seattle, WA) and hepatocyte antibody (OCH1E5, 1:20, Thermo Fisher, Waltham, MA) were then added to human liver slides in PBS/Tween, 3% goat serum at 250 µl per slide.

Techniques: Activity Assay, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: EBioMedicine

Article Title: Reduced circulating BMP10 and BMP9 and elevated endoglin are associated with disease severity, decompensation and pulmonary vascular syndromes in patients with cirrhosis

doi: 10.1016/j.ebiom.2020.102794

Figure Lengend Snippet: Liver BMP9 and BMP10 are present in hepatocytes and their expression levels are reduced in cirrhosis, whereas endoglin expression is increased . (A) Low power image (10X) of control and cirrhotic liver sections stained with haematoxylin and eosin (White scale bar length = 30 µm). (B–D) Total RNA was extracted from control ( n = 8) and cirrhotic ( n = 9) liver samples and analysed by qPCR for (B) BMP9 , (C) BMP10 and (D) ENG expression. Mann Whitney Test * P< 0.05. (E,F) 8 healthy human liver samples and 9 samples from patients with cirrhosis were stained with Hoescht nuclear stain (blue) and a hepatocyte-specific antibody (red) in addition to antibodies (green) for (E) BMP9 or (F) BMP10. Images were captured using confocal microscopy and 100 µm scale bars are shown.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies against BMP9 (AP2064A, 1:50 Abcepta, San Diego, CA), BMP10 (LS-C293026, 1:100, LSBio, Seattle, WA) and hepatocyte antibody (OCH1E5, 1:20, Thermo Fisher, Waltham, MA) were then added to human liver slides in PBS/Tween, 3% goat serum at 250 µl per slide.

Techniques: Expressing, Staining, MANN-WHITNEY, Confocal Microscopy