Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Up-regulated local BMP10 expression in the right atrium of precPH patients. ( A ) Quantification of the relative BMP10 mRNA expression in control ( n = 9) and precPH ( n = 5 CTEPH) RA tissues. ( B and C ) Quantification of total RA BMP10 fluorescent area and RA cardiomyocytes BMP10 intensity levels in control ( n = 6) and precPH ( n = 4 PAH) paraffin-embedded RA tissue sections stained against BMP10, respectively. Representative immunofluorescent stainings of BMP10, Ulex-rhodamine (Ulex, endothelium), and cardiac TroponinT (cTnT, myocardium) in the negative control sample for anti-rabbit Alexa488 and anti-mouse Alexa647 ( D ), in the control ( E ), and in the precPH ( F ) RA tissues at 60×-oil magnification. ( D’–F’ ) Alexa488 single-channel images from the stainings in ( D–F ). ( E ′′ and F′′ ) Zoom-in images from ( E′ and F′ ) to appreciate the sarcomeric pattern of the BMP10 staining in the cardiomyocytes and the homogeneous staining in the vessels. Scale bars = 50 μm. Brightness and contrast for the Alexa488 channel have not been modified. Normality of data was checked and transformed if needed, and statistical differences between precPH patients and controls were tested using an independent sample t -test.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Expressing, Control, Staining, Negative Control, Modification, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Up-regulated local BMP10 activity in the right atrium of precPH patients. ( A ) Quantification of positive pSMAD1/5/8 nuclei in vascular and non-vascular cells within the RA tissues from precPH ( n = 4 PAH) and controls ( n = 5). ( B and C ) Representative immunofluorescent staining of positive pSMAD1/5/8 nuclei in vascular and non-vascular cells from control and precPH with rhodamine and Alexa488 single-channel images on the sides. ( D ) Quantification of positive ID3 nuclei in vascular and non-vascular cells within the RA tissues from precPH ( n = 4 PAH) and controls ( n = 5). ( E and F ) Representative immunofluorescent staining of positive ID3 nuclei in vascular and non-vascular cells from control and precPH with rhodamine and Alexa488 single-channel images on the sides. Arrowheads indicate positive pSMAD1/5/8 and ID3 nuclei. Zoom-in images are included within ( B , C , E , and F ). Nuclei were counterstained with Hoechst 33342 and vessels with Ulex-rhodamine ( B , C , E , and F ). Negative control images are shown in , . Scale bars = 50 μm. Vascular and non-vascular measurements are plotted with their own Y -axis on the left or right side, respectively. Normality of data was checked and transformed if needed, and statistical differences between precPH patients and controls were tested using a Wilcoxon rank-sum test (in A and D ).

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Staining, Control, Negative Control, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Higher BMP10 plasma levels in precPH patients compared with controls. ( A and B ) BMP10 protein circulating plasma levels in precPH patients ( n = 48) and subgroups ( n = 48: 22 iPAH, 14 hPAH, and 12 CTEPH), respectively, vs. controls ( n = 16). ( C and D ) BMP9 protein circulating plasma levels in precPH patients ( n = 45) and subgroups ( n = 45: 20 iPAH, 14 hPAH, and 11 CTEPH), respectively, vs. controls ( n = 16). ( E and F ) Correlation between BMP10 and BMP9 plasma levels in precPH patients ( n = 45) or subgroups ( n = 45: 20 iPAH, 14 hPAH, and 11 CTEPH), respectively, vs. controls ( n = 16). Logarithmic Y -axis is used in graphs ( A – D ). Data in ( A and B ) are y + 1 for logarithmic scale transformation. Normality of data was checked and transformed if needed. Statistical differences between precPH patients or precPH subgroups and controls were tested with an independent sample t -test or a one-way ANOVA, respectively. Associations were tested with univariate linear regression analysis.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Clinical Proteomics, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: BMP10 transcriptional activity in precPH patients and controls. ( A ) Schematic explanation of the BRE-LUC reporter assay to determine BMP transcriptional activity in venous serum. Specific trap antibodies targeting BMP9 or BMP9 and BMP10 are used to assess BMP10 activity. Created with BioRender.com. B ) Relative BMP transcriptional activity as a luciferase read-out from the HMEC-BRE-LUC, endothelial cells expressing a BMP-specific luciferase reporter, in control ( n = 15) and precPH subgroups ( n = 21 iPAH, n = 13 hPAH, and n = 11 CTEPH) after incubation with phosphate-buffered saline (PBS) (baseline), anti-BMP9, or ALK1-Fc (inhibition of BMP9 and BMP10). ( C ) BMP10 activity in controls and precPH subgroups has been calculated from the subtraction of anti-BMP9 and ALK1-Fc to total BMP activity. Normality of data was checked and transformed if needed. Statistical differences between precPH patients and controls, and between baseline conditions and trap antibodies, were tested with an independent sample t -test or a one-way ANOVA, after which pairwise t -testing with Bonferroni correction was applied, respectively.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Reporter Assay, Luciferase, Expressing, Control, Incubation, Saline, Inhibition, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Patients with more right atrial dilatation, reduced RV ejection fraction, and higher NT-proBNP have higher levels of circulation BMP10 activity. ( A and B ) BMP10 transcriptional activity in precPH patients with RA or RV dilation, respectively. ( C–E ) BMP10 transcriptional activity in precPH patients with high RAP, reduced RVEF, or high NT-proBNP, respectively. PrecPH patients were stratified according to RA volume (>79 mL/mm 2 for male patients or >69 mL/mm 2 for female patients), RV end-diastolic volume index (≥109 mL/mm 2 for males, and ≥97 mL/mm 2 for females), RAP (>14 mmHg), RVEF (<35%), and NT-proBNP levels (>1100 ng/L). Normality of data was checked and transformed if needed. Statistical differences between both groups were tested with an independent samples t -test.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Effect of pressure unloading on BMP10 activity in precPH patients. ( A ) Serum relative BMP activity at baseline and post-PEA in CTEPH patients. Incubation with anti-BMP9 only blocked BMP9 activity, while ALK1-Fc blocked both BMP9 and BMP10 activities. ( B ) Calculated BMP10 transcriptional activity at baseline and post-PEA ( n = 13), respectively. BMP10 transcriptional activity is calculated by subtracting BMP activity values after incubation with the trap antibodies. Normality of data was checked and transformed if needed. Statistical differences between baseline conditions and trap antibodies, and between baseline and post-PEA, were tested with an independent sample t -test.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Incubation, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10

doi: 10.1074/jbc.m111.233577

Figure Lengend Snippet: FIGURE 1. In vivo processing of mouse pro-BMP10. A, right panel: Western blot of tissue extracts from RA and LA isolated from WT adult (3 months old) male mice using a mouse BMP10 Ab or anti-mouse HRP Ab (control). Left panel: ex vivo control, Western blot (WB) analysis of 20-h conditioned media fromCOS-1cellstransientlytransfectedwithnon-taggedpro-BMP10aloneor with furin using the same mouse BMP10 Ab. Proteins were resolved by 8% Tris-Tricine SDS-PAGE gels under non-reducing conditions. B, BMP10 mRNA levels were measured by QPCR in RA (dark gray bar) and LA from WT adult male mice. Mean S.D. are given and n 5 mice per group. Expression of BMP10 mRNA is restricted to adult mouse RA. C, PACE4, furin, and PC5/6 mRNA levels were determined by QPCR in RA (dark gray bars) and LA (light gray bars) from WT adult male mice. Mean S.D. are given and n 5 mice per group.NostatisticaldifferencewasobservedbetweenthemRNAlevelsofthe RA and LA.

Article Snippet: Western Blotting and Antibodies—Media from COS-1 cells (transfected with non-tagged pro-BMP10) or mouse atria protein extracts (50 g) were subjected to non-reducing (8% Tricine) SDS-PAGE analyses, followed by transfer to a 0.2- m PVDF membrane (Millipore) and BMP10 detection using a BMP10 antibody (BMP10 Ab; under non-reducing conditions) (1:500; R&D Systems) and the corresponding secondary antibody conjugated to horseradish peroxidase (HRP) (1:10,000; Invitrogen).

Techniques: In Vivo, Western Blot, Isolation, Control, Ex Vivo, SDS Page, Expressing

Journal: Journal of Biological Chemistry

Article Title: Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10

doi: 10.1074/jbc.m111.233577

Figure Lengend Snippet: FIGURE 2. In vitro, furin is better than PC5/6 or PACE4 at processing the 12-mer mouse BMP10 peptide at the predicted R311IRR3142 cleavage site, whereas PC7 does not cleave this peptide. A, typical RP-HPLC profile for in vitro digestion of the 12-mer mouse BMP10 peptide with soluble furin. The synthetic peptide (200 M) was incubated for 2 h in vitro with 2 units of purified soluble furin, PC5/6, PACE4, or PC7, as described under “Experimental Procedures.”TheproductswereseparatedbyRP-HPLConaVarianC18column(5m,100Å,4.6250mm).ThecleavagesiteRIRR2wasconfirmedbyMS/MS. The % cleavage was calculated as the ratio of the normalized peak areas (peak area/number of peptide bonds) of C-terminal fragment NAKG and the intact 12-mer peptide (at time 0). B, summary of the in vitro 2-h digestions of the 12-mer mBMP10 peptide with furin, PC5/6, PACE4, and PC7, respectively, based on RP-HPLC analyses. The results represent an average of two independent experiments. C, time dependent in vitro cleavage of the 12-mer mBMP10 peptide. The synthetic peptide was incubated with purified PCs in vitro for variable amounts of time. For each time point, the incubation mixture was subjected to RP-HPLC separation and the % cleavage was calculated and plotted as a function of time. Averages of two independent experiments are presented. Based on the linear range of the respective rate profiles (e.g. % cleavage at 20 min), the 12-mer mBMP10 peptide is a 3-fold better substrate for furin than for PC5/6 or PACE4.

Article Snippet: Western Blotting and Antibodies—Media from COS-1 cells (transfected with non-tagged pro-BMP10) or mouse atria protein extracts (50 g) were subjected to non-reducing (8% Tricine) SDS-PAGE analyses, followed by transfer to a 0.2- m PVDF membrane (Millipore) and BMP10 detection using a BMP10 antibody (BMP10 Ab; under non-reducing conditions) (1:500; R&D Systems) and the corresponding secondary antibody conjugated to horseradish peroxidase (HRP) (1:10,000; Invitrogen).

Techniques: In Vitro, Incubation, Purification

Journal: Journal of Biological Chemistry

Article Title: Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10

doi: 10.1074/jbc.m111.233577

Figure Lengend Snippet: FIGURE 3. Ex vivo validation of the R313IRR3162 cleavage site by site-di- rected mutagenesis. A, schematic representation of the 424-aa human pre- pro-BMP10 and its derived forms, pro-BMP10, prosegment, and mature BMP10 (BMP10). Depicted are the signal peptide (SP), N-terminal ProtC tag, potential N-glycosylation sites (N67, N131), predicted PC-processing site (R313IRR3162) and its mutants: P1 (R316A), P4 (R313A), and P1/P4 (R316A/ R313A). B, cell lysates (left) and 20-h conditioned media (right) from COS-1 cells transiently expressing (ProtC)-BMP10 carrying either no mutation (WT; lane 1) or mutations R316A (lane 2), R313A (lane 3), and R316A/R313A (lane 4), or (ProtC)-BMP10 WT and either prepro-furin (ppFurin; lane 5) or prepro- PACE4 (ppPACE4; lane 6), or no protein (vector, lane 7) were analyzed by West- ern blotting using a rabbit ProtC-Ab. Processing of pro-BMP10 (pro) WT into its prosegment is detected only in the medium (right). The mutant forms of pro-BMP10 (R316A, R313A, and R316A/R313A) are no longer cleaved. In cell lysates only the uncleaved form (pro-BMP10) is detected (left). *, nonspecific band. The data are representative of at least two independent experiments. WB, Western blot.

Article Snippet: Western Blotting and Antibodies—Media from COS-1 cells (transfected with non-tagged pro-BMP10) or mouse atria protein extracts (50 g) were subjected to non-reducing (8% Tricine) SDS-PAGE analyses, followed by transfer to a 0.2- m PVDF membrane (Millipore) and BMP10 detection using a BMP10 antibody (BMP10 Ab; under non-reducing conditions) (1:500; R&D Systems) and the corresponding secondary antibody conjugated to horseradish peroxidase (HRP) (1:10,000; Invitrogen).

Techniques: Ex Vivo, Biomarker Discovery, Mutagenesis, Derivative Assay, Glycoproteomics, Expressing, Plasmid Preparation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10

doi: 10.1074/jbc.m111.233577

Figure Lengend Snippet: FIGURE 4. Pro-BMP10 is cleaved ex vivo by endogenous furin (CHO-K1 cells), stably expressed furin (CHO-FD11/Fur cells), or transiently expressed furin (LoVo cells). Western blot (WB) analyses of 20-h condi- tionedmediumfromcells(HEK293,COS-1,CHO-K1,CHO-FD11/Fur,andCHO- FD11 cells) transiently transfected with either (ProtC)-BMP10 and an empty vector, or (ProtC)-BMP10 and the prosegment of furin (ppFurin), and from cells (LoVo cells) transfected with either empty vector, (ProtC)-BMP10 and emptyvector,(ProtC)-BMP10andfurin,or(ProtC)-BMP10andsFurin.The%of pro-BMP10 cleavage into its prosegment, calculated as prosegment/(pro- BMP10 prosegment) 100, is indicated below each lane along with the average % cleavage and S.D. values of several (n) independent experiments. The deficiency in endogenous PC activity of each cell line is depicted as PC. Note the lack of processing of pro-BMP10 overexpressed in cell lines deficient in endogenous furin activity (CHO-FD11 and LoVo cells).

Article Snippet: Western Blotting and Antibodies—Media from COS-1 cells (transfected with non-tagged pro-BMP10) or mouse atria protein extracts (50 g) were subjected to non-reducing (8% Tricine) SDS-PAGE analyses, followed by transfer to a 0.2- m PVDF membrane (Millipore) and BMP10 detection using a BMP10 antibody (BMP10 Ab; under non-reducing conditions) (1:500; R&D Systems) and the corresponding secondary antibody conjugated to horseradish peroxidase (HRP) (1:10,000; Invitrogen).

Techniques: Ex Vivo, Stable Transfection, Western Blot, Transfection, Plasmid Preparation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10

doi: 10.1074/jbc.m111.233577

Figure Lengend Snippet: FIGURE 5. Ex vivo processing of human pro-BMP10 by overexpressed furin, PACE4, and PC5/6. Western blot (WB) analyses of 20-h conditioned media from CHO-FD11 cells transiently transfected with either empty vector (vector; lane 8), or with a vector expressing ProtC-tagged pro-BMP10 (lanes 1–7; (ProtC)-BMP10) and vectors expressing either no protein (vector), furin, PACE4, or PC5/6, or their truncated versions sFurin, PACE4- C, or PC5/6- C. Proteins were revealed by using a rabbit ProtC-Ab. The corresponding per- centages of pro-BMP10 cleavage (%) calculated from the ratio of band inten- sities of prosegment/(pro-BMP10 prosegment) are indicated. The average % of pro-BMP10 cleavage and corresponding S.D. values from 4 independent experiments (n 4) are shown as a bar graph.

Article Snippet: Western Blotting and Antibodies—Media from COS-1 cells (transfected with non-tagged pro-BMP10) or mouse atria protein extracts (50 g) were subjected to non-reducing (8% Tricine) SDS-PAGE analyses, followed by transfer to a 0.2- m PVDF membrane (Millipore) and BMP10 detection using a BMP10 antibody (BMP10 Ab; under non-reducing conditions) (1:500; R&D Systems) and the corresponding secondary antibody conjugated to horseradish peroxidase (HRP) (1:10,000; Invitrogen).

Techniques: Ex Vivo, Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Journal of Biological Chemistry

Article Title: Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10

doi: 10.1074/jbc.m111.233577

Figure Lengend Snippet: FIGURE 6. Ex vivo processing of pro-BMP10 by the PCs and their deriva- tives, and inhibition by D6R and RVKR-cmk. A, Western blot (WB) analysis of 20-h conditioned medium from CHO-FD11 cells co-transfected with (ProtC)-BMP10 and either an empty vector or furin, sFurin, PACE4, PC5/6, or PC5/6- C.Asindicated,theconditionedmediumwascollectedafternotreat- ment (), or treatment () with either the cell permeable convertase inhibi- tor RVKR-cmk (25 M) or the cell surface convertase inhibitor D6R (10 M). For each condition, the average % of pro-BMP10 cleavage and corresponding S.D. values from three to four independent experiments are shown as a bar graph. B, Western blot analyses of 20-h conditioned medium from HEK293 cells (left) or COS-1 cells (right) transiently expressing (ProtC)-BMP10 or no protein (vector) and collected after no treatment (dimethyl sulfoxide, DMSO) or treatment with either the cell surface convertase inhibitor D6R (10 or 20 M) or the cell permeable convertase inhibitor RVKR-cmk (25 or 50 M). These data are representative of at least two independent experiments.

Article Snippet: Western Blotting and Antibodies—Media from COS-1 cells (transfected with non-tagged pro-BMP10) or mouse atria protein extracts (50 g) were subjected to non-reducing (8% Tricine) SDS-PAGE analyses, followed by transfer to a 0.2- m PVDF membrane (Millipore) and BMP10 detection using a BMP10 antibody (BMP10 Ab; under non-reducing conditions) (1:500; R&D Systems) and the corresponding secondary antibody conjugated to horseradish peroxidase (HRP) (1:10,000; Invitrogen).

Techniques: Ex Vivo, Inhibition, Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Journal of Biological Chemistry

Article Title: Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10

doi: 10.1074/jbc.m111.233577

Figure Lengend Snippet: FIGURE 7. Ex vivo, pro-BMP10 is processed in a post-medial Golgi com- partment (likely TGN) and is rapidly secreted into the media. A, HEK293 cells transiently transfected with (ProtC)-BMP10 were pulse-labeled with [35S]Met/Cys for 15 min and chased for 0, 30, 60, and 120 min in the absence oftheradiolabel.Celllysatesandmediumsampleswereimmunoprecipitated withamouseBMP10AbandthenresolvedbySDS-PAGE(8%Tris-Tricinegels) followed by autoradiography (4 days). B, HEK293 cells transiently transfected with nontagged pro-BMP10 (BMP10) were pulse-labeled with [35S]Met/Cys for 2 h in the absence of any treatment, or in the presence of brefeldin A (BFA; 2.5 g/ml), tunicamycin (Tun; 5 g/ml), D6R (10 M), RVKR-cmk (50 M), or dimethyl sulfoxide (DMSO). In control experiments HEK293 cells were tran- siently transfected with (ProtC)-BMP10 or no protein (vector). Cell lysates and medium samples were immunoprecipitated (IP) with mouse BMP10 Ab and then resolved by SDS-PAGE (8% Tris-Tricine gels) followed by autoradiogra- phy (17 h for pro-BMP10 detection and 4 days for detection of mature BMP10).

Article Snippet: Western Blotting and Antibodies—Media from COS-1 cells (transfected with non-tagged pro-BMP10) or mouse atria protein extracts (50 g) were subjected to non-reducing (8% Tricine) SDS-PAGE analyses, followed by transfer to a 0.2- m PVDF membrane (Millipore) and BMP10 detection using a BMP10 antibody (BMP10 Ab; under non-reducing conditions) (1:500; R&D Systems) and the corresponding secondary antibody conjugated to horseradish peroxidase (HRP) (1:10,000; Invitrogen).

Techniques: Ex Vivo, Transfection, Labeling, Autoradiography, Control, Plasmid Preparation, Immunoprecipitation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10

doi: 10.1074/jbc.m111.233577

Figure Lengend Snippet: FIGURE 8. Furin is the major pro-BMP10 cleaving enzyme in hepatocytes. Primary hepatocytes isolated from WT mice and those lacking PC5/6 (PC5/6- KO) or furin (Fur-KO) in hepatocytes (22) were transiently transfected with plasmids expressing no protein (vector) or (ProtC)-BMP10. Processing of pro- BMP10 into its prosegment was analyzed in 48-h conditioned medium by immunoprecipitation (IP) with a rabbit ProtC-Ab followed by Western blot- ting (WB) using the same antibody. The percentages (%) of cleavages in this particular experiment are indicated, along with the average % cleavage and S.D. values of two independent experiments.

Article Snippet: Western Blotting and Antibodies—Media from COS-1 cells (transfected with non-tagged pro-BMP10) or mouse atria protein extracts (50 g) were subjected to non-reducing (8% Tricine) SDS-PAGE analyses, followed by transfer to a 0.2- m PVDF membrane (Millipore) and BMP10 detection using a BMP10 antibody (BMP10 Ab; under non-reducing conditions) (1:500; R&D Systems) and the corresponding secondary antibody conjugated to horseradish peroxidase (HRP) (1:10,000; Invitrogen).

Techniques: Isolation, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Nature communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10.

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: Fig. 1 Overall structures of BMP10:BMPRII and ALK1:BMP10:BMPRII complexes. a One asymmetric unit of the BMP10:BMPRII 1.48 Å crystal structure. Chain identities (IDs) A to D are labelled. BMP10 is coloured in coral and light purple, BMPRII coloured in green. b 2.4 Å structure of BMP10:BMPRII with chain IDs labelled. Only one monomer of BMP10 (in coral) and BMPRII (in green) in an asymmetric unit. One symmetry-related molecule is shown in grey to illustrate the BMP10 dimer bound to two copies of BMPRII. c–e Overall structure of the ALK1:BMP10:BMPRII complex. Four copies of each BMP10, ALK1 and BMPRII monomers are found in one asymmetric unit, forming two copies of ternary signalling complexes shown in semi-transparent yellow and grey surface. Chain IDs in complex 1 (cpx1) (d) and cpx2 (e) are shown. In cpx1, BMP10 monomers are coloured in coral and light purple, ALK1 in yellow and BMPRII coloured in green. In cpx2, BMP10 monomers are coloured in coral and cyan, ALK1 in dark yellow, BMPRII in green. f An illustration of BMPRII- signalling complex in relation to cell surface. The last residues in ALK1 and BMPRII ECD cDNA-encoded sequences are 118 and 150, respectively. The last residues that can be seen in the crystal structures are shown in spheres and labelled. The 1.48 Å BMP10:BMPRII structure (in grey and semi-transparent) is superimposed on the ternary signalling complex (coloured as in Fig. 1d, cpx1) to show positions of further modelled sequence in BMPRII C-termini. The C-terminal 10–13 residues in both ALK1 and BMPRII that are not visible in the structure are represented by thick dashed lines.

Article Snippet: A high binding 96-well plate was coated with 0.25 μg/well of anti-human BMP10 GF-domain antibody (cat. No. MAB2926, R&D Systems) in phosphate-buffered saline (PBS) and incubated in a humidified chamber at 4 °C overnight.

Techniques: Sequencing

Journal: Nature communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10.

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: Fig. 2 Comparison of ALK1 binding sites in binary and ternary BMPRII receptor complexes. a Overlay of BMP10:ALK1 from the ternary signalling complex (cpx1, magenta, cpx2, purple) to those from binary complexes (PDB code 6SF1 in grey; 6SF3 in cyan for BMP10 and orange for ALK1). The backbones of all overlaid molecules are shown in ribbon. BMP10 from 6SF3 also shown in semi-transparent cartoon. Because in both 6SF1 and 6SF3, there was only one copy of BMP10:ALK1 monomer in an asymmetric unit, the dimeric receptor complexes for 6SF1 and 6SF3 were generated with a symmetry- related molecule and the two BMP10:ALK1 interfaces in 6SF1 and 6SF3 dimer would be identical. b Comparison of the buried surface area at the BMP10 and ALK1 interface in binary and ternary receptor complexes. c Overlay of all ALK1 chains, displayed in ribbon on BMP10 surface (light cyan). Four parts of BMP10 binding sites on ALK1 identified previously13 are highlighted by dashed lines. The colour for each chain is shown below. d Zoomed-in views of ALK1 H87 and E59 interaction area. H-bond interactions are shown with dotted lines. Same colour scheme as in c. The only interactions can be seen are in grey (from 6SF1), and orange and cyan (from 6SF3). Detailed information and the list of the interactions can be found in Supplementary Table 2.

Article Snippet: A high binding 96-well plate was coated with 0.25 μg/well of anti-human BMP10 GF-domain antibody (cat. No. MAB2926, R&D Systems) in phosphate-buffered saline (PBS) and incubated in a humidified chamber at 4 °C overnight.

Techniques: Comparison, Binding Assay, Generated

Journal: Nature communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10.

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: Fig. 3 Highly flexible BMP10:BMPRII interaction with a hinge in BMP10. a,b Secondary structural elements of the type II receptor (a, represented by BMPRII) or BMP (b, represented by BMP10), coloured in rainbow from blue at the N-terminus to red at the C-terminus. c, d Overlay of seven BMP10:BMPRII 1:1 complexes by BMPRII (c) and a zoomed-in view of the wrist helix region (d). e Overlay of seven BMP10:BMPRII 1:1 complexes by BMP10 wrist helix. In c–e, complex AI in green, CK in magenta, BJ in cyan, DL in dark yellow, AC in light purple, AB in dark blue, BD in grey. In c, e, red arrows highlight the movement of BMP10 among the 7 complexes, whilst the green arrow highlights the movement of BMPRII among the 7 complexes. f, g Overlay of complexes CK (magenta) and BD (grey) by BMP10 wrist helix. f. The hinge region and the distances between the BMPRII finger 2 hairpins and BMP10 fingertips 3/4 are shown. g A zoomed-in view of the hinge area, showing the rotation angle between complex CK V406/CA, complex BD K410/CA and complex BD V406/CA is 22.7 degrees.

Article Snippet: A high binding 96-well plate was coated with 0.25 μg/well of anti-human BMP10 GF-domain antibody (cat. No. MAB2926, R&D Systems) in phosphate-buffered saline (PBS) and incubated in a humidified chamber at 4 °C overnight.

Techniques:

Journal: Nature communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10.

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: Fig. 4 Detailed BMP10:BMPRII interface interactions. a BMP10:BMPRII binding interface in complex AC. BMP10 (in coral)-binding surface on BMPRII (purple) can be broadly divided into three regions. The central hydrophobic triad (Y67, W85 and F115), the β4 strand to the A-loop (green oval), the F3- loop and the region connecting the A-loop and the F3-loop (light blue circle and orange oval). b–f Detailed interactions between BMP10 and BMPRII, at the β4 strand and A-loop region in all complexes (b), at the F3-loop (c) and the regions connecting the A-loop and the F3-loop in complex AC (d), at BMPRII S107 region in complex BD (e) and AB (f). BMP10 is shown in coral sticks throughout b–f, whereas BMPRII is in grey for complex BD, in dark blue for complex AB, in yellow for complex DL, in magenta for complex CK, in cyan for complex BJ and in green for complex AI. In a–f red dashed lines denote H- bonds, with distance all between 2.7-3.7 Å if not labelled. Underlined residue numbers are those from BMP10, and residues numbers in normal text are those from BMPRII. g Buried interface area between BMP10 and BMPRII in binary and ternary receptor complexes. *Complex BD has much smaller buried surface area because some sidechains at the interface were deleted between the A-loop and F3-loop due to poor densities.

Article Snippet: A high binding 96-well plate was coated with 0.25 μg/well of anti-human BMP10 GF-domain antibody (cat. No. MAB2926, R&D Systems) in phosphate-buffered saline (PBS) and incubated in a humidified chamber at 4 °C overnight.

Techniques: Binding Assay, Residue

Journal: Nature communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10.

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: Fig. 5 Comparison of BMP10 binding site on BMPRII with BMP9 site on ActRIIB. a Sequence alignment of BMPRII ECD and ActRIIB ECD. Residues at the binding interface with BMP10 or BMP9 are shown in blue. Residues that are not modelled in the BMPRII structure are shown in grey. Hydrophobic triad residues are highlighted in cyan. Positions of the four loops are highlighted below the sequence. Lines above the sequence highlight the residues deleted in the mutagenesis studies. ΔF1(F3) = deletion of finger 1 (finger 3) residues; ΔGDP = deletion of residue Gly, Asp and Pro. b, c BMP10-binding site on BMPRII (b, light purple) and BMP9-binding site on ActRIIB (c, cyan). Residues making direct interactions with BMP10 or BMP9 are coloured in magenta, with hydrophobic triad residues shown in sticks. BMP10 G89 is highlighted in a magenta sphere. Water molecules that mediate hydrogen bond interactions between BMPRII and BMP10 are shown in yellow spheres. d BMPRII in binary (complex AC, purple cartoon) and ternary (complex AI, green cartoon) complexes on BMP10 surface (pale blue), showing G89 (magenta spheres) docking into a deep pocket on BMP10. e ActRIIB (cyan cartoon) on BMP9 surface (pale blue, from PDB entry 4FAO) does not have A-loop mediated interaction. In d, e, residues making H-bond interactions shown in magenta, and hydrophobic triad shown in magenta sticks.

Article Snippet: A high binding 96-well plate was coated with 0.25 μg/well of anti-human BMP10 GF-domain antibody (cat. No. MAB2926, R&D Systems) in phosphate-buffered saline (PBS) and incubated in a humidified chamber at 4 °C overnight.

Techniques: Comparison, Binding Assay, Sequencing, Mutagenesis, Residue

Journal: Nature communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10.

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: Fig. 6 Context-dependent fingertip 3/4 conformation in BMP9 and BMP10. a An overlay of all BMP10 monomers from different protein interaction contexts. Sources of structures are listed in Supplementary Table 5. b A close-up view of fingertip 3/4 with all the structures annotated. c A closed-up view of fingertip 3/4 region after superposition of all BMP9 monomers from different protein interaction contexts. Sources of the structures are listed in Supplementary Table 5. The overlay of the full monomers can be found in Supplementary Fig 9. d A schematic diagram depicting the context-dependent conformation of fingertip 3/4 in BMP9 and BMP10.

Article Snippet: A high binding 96-well plate was coated with 0.25 μg/well of anti-human BMP10 GF-domain antibody (cat. No. MAB2926, R&D Systems) in phosphate-buffered saline (PBS) and incubated in a humidified chamber at 4 °C overnight.

Techniques:

Journal: Nature communications

Article Title: Crystal structures of BMPRII extracellular domain in binary and ternary receptor complexes with BMP10.

doi: 10.1038/s41467-022-30111-2

Figure Lengend Snippet: Fig. 8 BMP10:BMPRII interaction is important for prodomain displacement. a A representative native PAGE of prodomain displacement experiment. b Quantification of native PAGE results. The band intensities of Pro:BMP10 and prodomain were obtained by densitometry using ImageJ. The ratio of Pro:BMP10/prodomain for each lane was calculated, then normalised to that of the WT control run on the same gel. Each mutant protein was run on at least three independent native PAGE experiments, with two WT controls on each gel to allow normalisation. Final N numbers for each sample are labelled below the graph. Conditions with BMPRII ECDs at 2-fold excess (2x) are shown in open bars, and those with 5x in hashed bars. The two dotted lines mark the mean values for 2x and 5x WT BMPRII, respectively. Data are presented as mean values + /−SEM. One-way ANOVA analysis for WT ECD group, ****P < 0.0001, **P < 0.01; for BMPRII at 2x excess group, Dunnett’s post test comparing with WT BMPRII ECD, $P < 0.05, $$P < 0.01; for BMPRII at 5x excess group, Dunnett’s post test comparing with WT BMPRII ECD, #P < 0.05, ###P < 0.001, ####P < 0.0001. c Schematic depicting the experiment of the prodomain displacement by ELISA. d Quantitative results of prodomain displacement by ELISA. All readings are normalised to the WT on the same plate. N = 5 for WT ECD, N = 3 for each mutant protein. Each N number represents an independent ELISA experiment. Data are presented as mean values + /− SEM. One-way ANOVA analysis for BMPRII at 125x excess, with Dunnett’s post test comparing with WT BMPRII ECD (mean value shown as a dotted line). #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001. In b, d, coloured in red are residues near the hydrophobic triad, in orange are residues from the A- loop, in yellow are F3-loop; in brown, non-interface residues, and in purple, F1-loop which is not visible in the structure. Source data, including the exact p values for b, d, are provided as a Source Data file. ECD = extracellular domain, GF-domain = growth factor domain, Pro:BMP10 = non-covalent complex of BMP10 prodomain with its GF-domain.

Article Snippet: A high binding 96-well plate was coated with 0.25 μg/well of anti-human BMP10 GF-domain antibody (cat. No. MAB2926, R&D Systems) in phosphate-buffered saline (PBS) and incubated in a humidified chamber at 4 °C overnight.

Techniques: Clear Native PAGE, Control, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction

doi: 10.1172/jci.insight.125908

Figure Lengend Snippet: Real-time qPCR analysis of Notch pathway genes and their downstream targets using RNA isolated from E10.5, E12.5, and E14.5 control and Plxnd1–/– hearts (A). Expression levels of Nrg1, ErbB2, and ErbB4, as measured by qPCR using RNA isolated from E10.5, E12.5, and E14.5 control and Plxnd1–/– hearts. n = 3 for each genotype/time point (B). Representative Western blot of Notch1 and NICD on E12.5 and E14.5 control and Plxnd1–/– heart lysates. Gapdh was used as loading control (C). Immunostaining for NICD on E14.5 control and Plxnd1–/– heart sections (D). Representative Western blot analysis of ErbB2 and pErbB2 using E12.5 and E14.5 control and Plxnd1–/– heart lysates. Actin was used as loading control (E). Partial rescue of hypertrabeculation and noncompaction defects displayed by Plxnd1–/– embryos after treatment with the γ-secretase inhibitor DBZ. H&E staining of E13.5 control and Plxnd1–/– hearts isolated after vehicle or DBZ treatment (F). Quantification of the thickness of the compact and trabecular layer (G). n = 3 for each genotype. Real-time qPCR analysis of Notch1, Bmp10, and Adamts15 using RNA isolated from E13.5 control and Plxnd1–/– hearts isolated after vehicle or DBZ treatment (H). LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Scale bars: 100 μm.

Article Snippet: The following antibodies were used for Western blotting and immunohistochemistry: Notch1 (Santa Cruz, catalog sc-6014), Cleaved Notch1 (Val1744) (Cell Signaling, catalog 4147S), ErbB2 (Cell Signaling, catalog 2242), pErbB2 (Cell Signaling, catalog 2247), Bmp10 (R&D System, catalog MAB6038), Vinculin (MilliporeSigma, catalog V9131), Gapdh (Santa Cruz, catalog sc-20357), Actin (Santa Cruz, catalog sc-47778), MF20-C (DSHB), Ki-67 (Abcam, catalog ab15580), Sema3E (MilliporeSigma, catalog HPA029419), Varsican (Thermo Fisher Scientific, catalog PA11748A), Adamts1 (Santa Cruz, catalog sc-47727), Fibronectin (Santa Cruz, catalog sc-8422), and PlexinD1 (R&D System, catalog AF4160).

Techniques: Isolation, Control, Expressing, Western Blot, Immunostaining, Staining

Journal: Angiogenesis

Article Title: BMP9 knockout impairs pulmonary vessel muscularisation and confers aberrant tamoxifen sensitivity

doi: 10.1007/s10456-025-10017-5

Figure Lengend Snippet: RNA sequencing of Gdf2 −/− lungs identifies genes associated with BMP9 loss. a RNA was isolated from wild type (WT; n = 4) and Bmp9 KO (n = 6) mice lungs. Following RNA libraries preparation, samples were analysed for 50 bp paired end reads on a Novaseq 6000 sequencer (Illumina). Volcano plot of differentially expressed genes in Bmp9 KO versus WT after fitting linear models and adjusting P values for multiple testing. b Schematic of treatment regime. WT and Bmp9 KO mice were administered daily for 3-weeks with 0.03 mg/kg recombinant human BMP9 or vehicle control. Mice were bled at the beginning and end of treatment regime to check BMP9 levels. c Lung sections were immunostained with α-smooth muscle actin (αSMA). Quantification of non-, partially, or fully-muscularised vessels as a percentage of arteries associated with alveolar ducts in WT (n = 11), Bmp9 KO plus vehicle (n = 7) and Bmp9 KO plus BMP9 (n = 8) mice. 20 arteries were counted per animal. d RNA was isolated from WT (n = 11), Bmp9 KO plus vehicle (n = 6/7) and Bmp9 KO plus BMP9 (n = 6/8) mice lungs. Gene expression Anxa8 , Colq , Dnah1 , Itga6 , Rbp3 , Syt15 and Tgtp1 was normalised against the housekeeping gene, Hprt . e and f Human pulmonary microvascular cells (PMVECs; n = 4 biological replicates) were serum-starved (0.1%) overnight prior to treatment with BMP9 or BMP10 (0.1, 0.3, 1 ng/ml) for 8 h. Gene expression of ITGA6 ( e ) and SYT15 ( f ) was measured using qPCR, normalised to 2 housekeeping genes ( B2M and HPRT ). ( c ) Two-way ANOVA. ( d , e, and f ) One-way ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± S.E.M

Article Snippet: After washing, biotinylated anti‐human BMP10 detection antibody (0.1 μg/well; R&D Systems) was added in PBS/1% BSA containing 0.2% GS.

Techniques: RNA Sequencing, Isolation, Recombinant, Control, Gene Expression

Journal: Angiogenesis

Article Title: BMP9 knockout impairs pulmonary vessel muscularisation and confers aberrant tamoxifen sensitivity

doi: 10.1007/s10456-025-10017-5

Figure Lengend Snippet: Bmp9 KO and double knockout mice treated with tamoxifen exhibit reduced smooth muscle associated gene expression. a Schematic of treatment regime. Bmp10 fl/fl (WT), Bmp10 fl/fl x Gdf2 −/− ( Bmp9 KO), Bmp10 fl/fl xRosa26 Cre−ERT ( Bmp10 cKO) and Bmp10 fl/fl xRosa26 Cre−ERT x Gdf2 −/− (dKO) mice were treated with tamoxifen once a day for five days with a two-day recovery period followed by a further 5 days at a dose of 40 mg/kg. As a vehicle control, WT mice were administered corn oil for the same period. Mice then underwent right heart catheterisation on day 56. Mice were also bled at day -3, 21 and 56 to assess BMP9 levels. Right atrium was also taken at day 56 to generate BMP10 conditioned media (RACM). Relevant tissue was collected on day 56. b–l RNA was isolated on day 56 from lungs of WT (corn oil; n = 8), WT (tamoxifen; n = 8), Bmp9 KO (tamoxifen; n = 8), Bmp10 cKO (tamoxifen; n = 8) and dKO (tamoxifen; n = 8). Gene expression was normalised against the housekeeping gene, Hprt . Acta2 ( b ), Des ( c ), Myh11 ( d ), Anxa8 ( e ), Colq ( f ), Rbp3 ( g ), Itga6 ( h ), Tgtp1 ( i ), Syt15 ( j ), Bmpr2 ( k ), Eng ( l ) and Smad6 ( m ) gene expression. b – m One-way ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± S.E.M

Article Snippet: After washing, biotinylated anti‐human BMP10 detection antibody (0.1 μg/well; R&D Systems) was added in PBS/1% BSA containing 0.2% GS.

Techniques: Double Knockout, Gene Expression, Control, Isolation

Journal: Angiogenesis

Article Title: BMP9 knockout impairs pulmonary vessel muscularisation and confers aberrant tamoxifen sensitivity

doi: 10.1007/s10456-025-10017-5

Figure Lengend Snippet: Transcriptional changes in conditional knockout mice treated with anti-BMP9. a Schematic of treatment regime. Bmp10 fl/fl xRosa26 Cre−ERT ( Bmp10 -cKO) were treated with tamoxifen once a day for five days with a two-day recovery period followed by a further 5 days at a dose of 40 mg/kg. As a vehicle control Bmp10 fl/fl (WT) mice were administered corn oil for the same period. On day 21 mice were dosed weekly for 2-weeks with 5mg/kg BMP9 antibody (anti-BMP9) or equivalent volume of mouse IgG2B (IgG) isotype as a control. Mice then underwent right heart catheterisation on day 42. Mice were also bled at day -3, 21 and 42 to assess BMP9 levels. Right atrium was also taken at day 42 to generate BMP10 conditioned media (RACM). Relevant tissue was collected on day 42. b Conditioned media from right atria collected at day 42 from WT (corn oil; n = 6), Bmp10 cKO – IgG (tamoxifen; n = 3) and Bmp10 cKO—anti-BMP9 (tamoxifen; n = 3) mice was assayed for BMP10 levels using a BMP10 growth factor domain (GFD) specific ELISA. c Serum from WT (corn oil; n = 6), Bmp10 cKO—IgG (n = 9) and Bmp10 cKO—anti-BMP9 ( Bmp10 cKO—anti-BMP9; n = 9) mice bled at day -3, 21 and 42 were assayed for BMP9 levels using a BMP9 specific ELISA. d – l RNA was isolated on day 45 from lungs of Bmp10 cKO (IgG; n = 8) and Bmp10 cKO (anti-BMP9; n = 9). Gene expression was normalised against the housekeeping gene, Hprt . Anxa8 ( d ), Colq ( e ), Dnah1 ( f ), Itga6 ( g ) Syt15 ( h ), Tgtp1 ( i ), Edn1 ( j ), Adm ( k ) and Smad6 ( l ) expression. ( e , g , h , i , j , k, and l ) Unpaired t-test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± S.E.M

Article Snippet: After washing, biotinylated anti‐human BMP10 detection antibody (0.1 μg/well; R&D Systems) was added in PBS/1% BSA containing 0.2% GS.

Techniques: Knock-Out, Control, Enzyme-linked Immunosorbent Assay, Isolation, Gene Expression, Expressing

Journal: Angiogenesis

Article Title: BMP9 knockout impairs pulmonary vessel muscularisation and confers aberrant tamoxifen sensitivity

doi: 10.1007/s10456-025-10017-5

Figure Lengend Snippet: Bmp9 KO and double knockout mice treated with tamoxifen exhibit extensive tissue remodelling. ( a – g ) Bmp10 fl/fl (WT), Bmp10 fl/fl x Gdf2 −/− ( Bmp9 KO), Bmp10 fl/fl xRosa26 Cre−ERT ( Bmp10 cKO) and Bmp10 fl/fl xRosa26 Cre−ERT x Gdf2 −/− (dKO) mice were treated with tamoxifen once a day for five days with a two-day recovery period followed by a further 5 days at a dose of 40 mg/kg. As a vehicle control, WT mice were administered corn oil for the same period. Mice then underwent right heart catheterisation on day 56. Relevant tissue was collected on day 56. a Heart weight was assessed as a ratio of femur length in WT (corn oil; n = 12), WT (tamoxifen; n = 20), Bmp9 KO (tamoxifen; n = 20), Bmp10 cKO (tamoxifen; n = 15) and dKO (tamoxifen; n = 13). b Spleen weight was assessed as a ratio of femur length in WT (corn oil; n = 12), Bmp10 fl/fl (tamoxifen; n = 20), Bmp9 KO (tamoxifen; n = 20), Bmp10 cKO (tamoxifen; n = 15), and dKO (tamoxifen; n = 13). c Ratio of right ventricle (RV) thickness and left ventricle thickness (LV) in WT (corn oil; n = 6), WT (tamoxifen; n = 10), Bmp9 KO (tamoxifen; n = 8), Bmp10 cKO (tamoxifen; n = 8) and dKO (tamoxifen; n = 6). d Heart rate was measured in WT (corn oil; n = 12), WT (tamoxifen; n = 20), Bmp9 KO (tamoxifen; n = 20), Bmp10 cKO (tamoxifen; n = 14) and dKO (tamoxifen; n = 13). e Measurement of cardiac output in WT (corn oil; n = 12), WT (tamoxifen; n = 19), Bmp9 KO (tamoxifen; n = 19), Bmp10 cKO (tamoxifen; n = 13) and dKO (tamoxifen; n = 10). f Right ventricular systolic pressure (RVSP) was measured in WT (corn oil; n = 12), WT (tamoxifen; n = 20), Bmp9 KO (tamoxifen; n = 20), Bmp10 cKO (tamoxifen; n = 14) and dKO (tamoxifen; n = 13). g Lung sections were immunostained with α-smooth muscle actin (αSMA). Quantification of non, partially, or fully-muscularised vessels as a percentage of arteries associated with alveolar ducts in WT (corn oil; n = 12), WT (tamoxifen; n = 15), Bmp9 KO (tamoxifen; n = 15), Bmp10 -cKO (tamoxifen; n = 15) and dKO (tamoxifen; n = 13) mice. 20 arteries were counted per animal. h Alveoli area was assessed in haematoxylin and eosin-stained lung sections. Percentage of counterstained tissue versus no staining of the whole lung area in WT (corn oil; n = 12), WT (tamoxifen; n = 15), Bmp9 KO (tamoxifen; n = 15), Bmp10 -cKO (tamoxifen; n = 15) and dKO (tamoxifen; n = 13) mice. ( i ) Lung sections were stained with Perl’s iron stain. Percentage of Perl’s positive cells in whole lung area from WT (corn oil; n = 12), WT (tamoxifen; n = 15), Bmp9 KO (tamoxifen; n = 15), Bmp10 -cKO (tamoxifen; n = 15) and dKO (tamoxifen; n = 13) mice. Scale bar = 100 μm. ( a , b , c , d , e , f , h and i ) One-way ANOVA. g Two-way ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Error bars represent mean ± S.E.M

Article Snippet: After washing, biotinylated anti‐human BMP10 detection antibody (0.1 μg/well; R&D Systems) was added in PBS/1% BSA containing 0.2% GS.

Techniques: Double Knockout, Control, Staining

Journal: Angiogenesis

Article Title: BMP9 knockout impairs pulmonary vessel muscularisation and confers aberrant tamoxifen sensitivity

doi: 10.1007/s10456-025-10017-5

Figure Lengend Snippet: Bmp9 KO mice treated with tamoxifen have cardiomegaly and splenomegaly. Wild type (WT; n = 9), Bmp9 KO (n = 7) and Bmp10 fl/fl xRosa26 Cre−ERT ( Bmp10 cKO; n = 2) mice were treated with tamoxifen once a day for five days with a two-day recovery period followed by a further 5 days at a dose of 40 mg/kg. Mice were also bled at day -3, 21 and 42 to assess BMP9 levels. Right atria were collected at day 42 for conditioned media culture. a Serum from WT (n = 8), Bmp9 KO (n = 7) and Bmp10 cKO (n = 2) mice bled at day -3, 21 and 42 were assayed for BMP9 levels using a BMP9 specific ELISA. BMP9 was undetectable in Bmp9 KO mice. b Conditioned media from right atria collected at day 42 from WT (n = 8), Bmp9 KO (n = 7) and Bmp10 cKO (n = 2) mice were assayed for BMP10 levels using a BMP10 growth factor domain (GFD) specific ELISA. c Heart weight was assessed as a ratio of femur length in WT (n = 9), Bmp9 KO (n = 7) and Bmp10 -cKO (n = 2). d Spleen weight was assessed as a ratio of femur length in WT (n = 9), Bmp9 KO (n = 7) and Bmp10 cKO (n = 2). e Alveoli area was assessed using haematoxylin and eosin and lung sections were stained with Perl’s iron stain. Scale bar = 100 μm. f Percentage of Perl’s positive cells in whole lung area from WT (n = 9), Bmp9 KO (n = 7) and Bmp10 -cKO (n = 2) mice. b , c, and d One-way ANOVA. f Unpaired t-test. * P ≤ 0.05, ** P ≤ 0.01. Error bars represent mean ± S.E.M

Article Snippet: After washing, biotinylated anti‐human BMP10 detection antibody (0.1 μg/well; R&D Systems) was added in PBS/1% BSA containing 0.2% GS.

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: The Prodomain-bound Form of Bone Morphogenetic Protein 10 Is Biologically Active on Endothelial Cells *

doi: 10.1074/jbc.M115.683292

Figure Lengend Snippet: BMP10 prodomain inhibits BMP10 activity in C2C12 cells. A , titration of BMP10 GFD activity in C2C12 cells. Serum-starved C2C12 cells were treated with BMP10 GFD at increasing concentrations for 1 h, and the phosphorylation of Smad1/5 and the induction of ID1 and ID3 proteins were measured by immunoblotting analysis. Total Smad1 was used as a loading control. B , prodomain inhibition assay in C2C12 cells. BMP10 GFD was pre-incubated with the prodomain (molar ratio BMP10 GFD to prodomain, 1:0, 1:1, 1:4, 1:16, and 1:64) before applying to the serum-starved C2C12 cells for 1 h. Ratio 0:64 indicates the same amount of prodomain as in 1:64, but in the absence of BMP10 GFD. The remaining activity was measured by phosphorylation of Smad1/5 using immunoblotting; total Smad1 was used as a loading control. One representative blot from four repeats is shown. Below, using densitometry analysis (Image J), relative phosphorylation of Smad1/5 were corrected to total Smad1 and normalized to the sample treated with BMP10 only without the prodomain (1:0). The fold changes are expressed as mean ± S.E. n = 4; ****, p ≤ 0.0001; n.s., not significant. One-way ANOVA, Dunnett's post test.

Article Snippet: Anti-BMP9 antibody (MAB3209), anti-BMP10 antibody (MAB2926), BMP10 prodomain (3956-BP-050), anti-BMP10 propeptide antibody (AF3956), biotinylated anti-BMP10 propeptide antibody (BAF3956), ALK1-Fc (370-AL), human BMP10 GFD (2926-BP-025) were all purchased from R&D Systems, Inc. Anti-phosphoSmad1/5/8 and anti-phosphoSmad1/5 antibody were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Titration, Phospho-proteomics, Western Blot, Control, Inhibition, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The Prodomain-bound Form of Bone Morphogenetic Protein 10 Is Biologically Active on Endothelial Cells *

doi: 10.1074/jbc.M115.683292

Figure Lengend Snippet: BMP10 prodomain does not inhibit BMP10 activity in endothelial cell lines. A , titration of BMP10 GFD activities in HPAECs ( left ) and HAECs ( right ). Increasing concentrations of BMP10 GFD as indicated were used to treat the serum-starved cells. After 1 h treatment, cells were harvested and the phosphorylation of Smad1/5 and the induction of ID1 and ID3 proteins were analyzed using immunoblotting. B , BMP10 GFD was pre-incubated with increasing amounts of BMP10 prodomain in same molar ratio as in B before applying to serum-starved endothelial cells in ( B ) HPAECs, ( C ) HAECs, and ( D ) HMEC-1. Remaining activity of BMP10 was measured by phosphorylation of Smad1/5 with total Smad1 as a loading control. One representative blot from four repeats is shown for all experiments. Prodomain inhibitions were quantified and analyzed as in B and shown below.

Article Snippet: Anti-BMP9 antibody (MAB3209), anti-BMP10 antibody (MAB2926), BMP10 prodomain (3956-BP-050), anti-BMP10 propeptide antibody (AF3956), biotinylated anti-BMP10 propeptide antibody (BAF3956), ALK1-Fc (370-AL), human BMP10 GFD (2926-BP-025) were all purchased from R&D Systems, Inc. Anti-phosphoSmad1/5/8 and anti-phosphoSmad1/5 antibody were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Titration, Phospho-proteomics, Western Blot, Incubation, Control

Journal: The Journal of Biological Chemistry

Article Title: The Prodomain-bound Form of Bone Morphogenetic Protein 10 Is Biologically Active on Endothelial Cells *

doi: 10.1074/jbc.M115.683292

Figure Lengend Snippet: Generation of recombinant human pBMP10. A , schematic diagram of BMP10 production and processing. B , FPLC chromatography gel filtration trace of purified pBMP10. C , purified pBMP10 shown as the peak fraction from the gel filtration in B on a non-reducing SDS-PAGE. A single asterisk denotes a nonspecific protein. D , both D- and M-forms of BMP10 GFD could be detected by monoclonal anti-BMP10 antibody. E, prodomain can be detected by anti-BMP10 prodomain antibody. BMP10 GFD from R&D Systems was used as a positive control in D and negative control in E .

Article Snippet: Anti-BMP9 antibody (MAB3209), anti-BMP10 antibody (MAB2926), BMP10 prodomain (3956-BP-050), anti-BMP10 propeptide antibody (AF3956), biotinylated anti-BMP10 propeptide antibody (BAF3956), ALK1-Fc (370-AL), human BMP10 GFD (2926-BP-025) were all purchased from R&D Systems, Inc. Anti-phosphoSmad1/5/8 and anti-phosphoSmad1/5 antibody were purchased from Cell Signaling Technology.

Techniques: Recombinant, Chromatography, Filtration, Purification, SDS Page, Positive Control, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The Prodomain-bound Form of Bone Morphogenetic Protein 10 Is Biologically Active on Endothelial Cells *

doi: 10.1074/jbc.M115.683292

Figure Lengend Snippet: Prodomain-bound BMP10 is a highly stable complex. Analytical gel filtration analysis of semi-purified pBMP10 in TBS ( A ), TBS with NaCl at 1 m final concentration ( B ) or TBS with 1 m GuHCl ( C ). pBMP10 was pre-incubated in each buffer for 30 min before being loaded onto a Superdex S200 10/300 size-exclusion column pre-equilibrated in the same buffer. A control protein carbonic anhydrase (24 kDa) that does not interact with either BMP10 GFD or its prodomain was added to the pBMP10 before loading. Blue dextran (2000 kDa, black arrow ) was run separately in each buffer to indicate the void volume. Note the protein peaks shifted slightly between the runs, potentially due to proteins interacting with the column matrix differently in different buffer systems. Points 1–11 on the traces correspond to consecutive fractions, which were run in lanes 1–11 of immunoblotting analyses probed with either anti-BMP10 prodomain or anti-BMP10 antibodies. Peaks X and Y were TCA precipitated, ran on a non-reducing SDS-PAGE and Coomassie Blue stained to reveal the identity of the peaks. Peak Z is carbonic anhydrase.

Article Snippet: Anti-BMP9 antibody (MAB3209), anti-BMP10 antibody (MAB2926), BMP10 prodomain (3956-BP-050), anti-BMP10 propeptide antibody (AF3956), biotinylated anti-BMP10 propeptide antibody (BAF3956), ALK1-Fc (370-AL), human BMP10 GFD (2926-BP-025) were all purchased from R&D Systems, Inc. Anti-phosphoSmad1/5/8 and anti-phosphoSmad1/5 antibody were purchased from Cell Signaling Technology.

Techniques: Filtration, Purification, Concentration Assay, Incubation, Control, Western Blot, SDS Page, Staining

Journal: The Journal of Biological Chemistry

Article Title: The Prodomain-bound Form of Bone Morphogenetic Protein 10 Is Biologically Active on Endothelial Cells *

doi: 10.1074/jbc.M115.683292

Figure Lengend Snippet: Prodomain-bound BMP10 is active in endothelial cells. A and B, phosphorylation of Smad1/5 in HPAECs ( A ) and HAECs ( B ) treated with 0.1 ng/ml, 0.33 ng/ml, and 1 ng/ml of BMP10 GFD or three independent preparations of pBMP10 for 1 h, detected by Smad1/5 phosphorylation in immunoblotting analysis; total Smad1 was used as a loading control. The concentrations of pBMP10 in all the cell assays refer to the concentrations of mature GFD in the pBMP10 complex. Relative phosphorylation of Smad1/5 upon treatment was measured using densitometry, corrected to total Smad1 and normalized to 0.1 ng/ml BMP10 GFD treatment condition and plotted on the right. C, induction of ID1 and BMPR2 mRNA expression by recombinant pBMP10, compared with BMP9 and BMP10 GFD. HPAECs were treated with the ligands at indicated concentrations for 8 h before samples were harvested for RNA extraction and qPCR analysis as described in “Experimental Procedures.” n = 3; *, p ≤ 0.05; n.s. , not significant; paired t test.

Article Snippet: Anti-BMP9 antibody (MAB3209), anti-BMP10 antibody (MAB2926), BMP10 prodomain (3956-BP-050), anti-BMP10 propeptide antibody (AF3956), biotinylated anti-BMP10 propeptide antibody (BAF3956), ALK1-Fc (370-AL), human BMP10 GFD (2926-BP-025) were all purchased from R&D Systems, Inc. Anti-phosphoSmad1/5/8 and anti-phosphoSmad1/5 antibody were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Western Blot, Control, Expressing, Recombinant, RNA Extraction

Journal: The Journal of Biological Chemistry

Article Title: The Prodomain-bound Form of Bone Morphogenetic Protein 10 Is Biologically Active on Endothelial Cells *

doi: 10.1074/jbc.M115.683292

Figure Lengend Snippet: BMPR-II ECD can release BMP10 GFD from the pBMP10 complex. A and B , interaction between BMPR-II ECD and pBMP10 was investigated using native PAGE. Purified pBMP10 complex was run on a 10% native PAGE ( A ) either alone (1:0), or with increasing amounts of BMPR-II ECD (molar ratio of pBMP10:BMPR-II ECD, 1:1, 1:2, and 1:4). 0:1 refers to BMPR-II alone control. Prodomain-bound BMP10 complex was separated into three bands on the native PAGE. These three bands were excised from the native PAGE, run in parallel on non-reducing SDS-PAGE, and probed with either anti-BMP10 antibody or anti-BMP10 prodomain antibody ( B ). Band 1 , which ran as a smeared band and stayed in the stacking gel, contains only BMP10 GFD, consistent with the PI of BMP10 GFD being 8.67. Band 3 contained only the prodomain (PI = 4.54) and ran fastest on the SDS-PAGE. Band 2 contained both BMP10 GFD and the prodomain, hence is the pBMP10 complex. The arrow in A points to the decrease in the pBMP10 complex upon adding increasing amounts of BMPR-II ECD. A single asterisk in B denotes either a nonspecific protein in the protein prep or a differentially processed BMP10 prodomain. C , changes of pBMP10 complex in A were quantified as the ratio of the pBMP10 complex/prodomain alone to normalize for the loading. n = 4, one-way ANOVA, Dunnett's post test. **, p ≤ 0.01.

Article Snippet: Anti-BMP9 antibody (MAB3209), anti-BMP10 antibody (MAB2926), BMP10 prodomain (3956-BP-050), anti-BMP10 propeptide antibody (AF3956), biotinylated anti-BMP10 propeptide antibody (BAF3956), ALK1-Fc (370-AL), human BMP10 GFD (2926-BP-025) were all purchased from R&D Systems, Inc. Anti-phosphoSmad1/5/8 and anti-phosphoSmad1/5 antibody were purchased from Cell Signaling Technology.

Techniques: Clear Native PAGE, Purification, Control, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: The Prodomain-bound Form of Bone Morphogenetic Protein 10 Is Biologically Active on Endothelial Cells *

doi: 10.1074/jbc.M115.683292

Figure Lengend Snippet: BMP10 derived from atrium or plasma is fully active. A , BMP10 mRNA expression in human heart tissues. n = 3. **, p ≤ 0.01; one-way ANOVA, Tukey's post test. B , BMP10 mRNA expression in mouse heart tissues. BMP10 expression is significantly higher in RA than LA in mouse. n = 4, paired t test; **, p ≤ 0.01; C , BMP activity could be detected in the conditioned medium of cultured mouse RA. Conditioned medium from LA or RA was applied to serum-starved HPAECs (both at 5% v / v ), and the BMP activity was measured by the induction of ID1 gene expression. No BMP activity can be detected from LA-conditioned medium ( LA CM ), while significant level of ID1 gene induction activity can be detected in the RA-conditioned medium ( RA CM ). n = 3, * p ≤ 0.05; n.s., not significant. One-way ANOVA, Tukey's post test; D , identification of BMP activity in RA CM. The ID1 -induction activity from RA CM (0.4% v / v ) could not be inhibited by anti-BMP9 antibody (at 20 μg/ml), but can be partially inhibited by anti-BMP10 antibody (at 20 μg/ml), and very effectively inhibited by ALK1-Fc (at 2.5 μg/ml). n = 3, *, p ≤ 0.05; n.s ., not significant. One-way ANOVA, Dunnett's post test; E , control experiments showed that anti-BMP9 antibody (at 10 μg/ml) could specifically neutralize BMP9 activity very effectively, but not the activity of BMP10 or pBMP10, whereas ALK1-Fc (at 2.5 μg/ml) can inhibit both BMP9 and BMP10 activity very effectively. The concentrations of BMP9, BMP10, and pBMP10 used in this assay were all 1 ng/ml. n = 3, one-way ANOVA for each BMP ligand group, Dunnett's post test, **, p ≤ 0.01; *, p ≤ 0.05; n.s ., not significant; F , BMP10 activity can be detected in human plasma. Freshly frozen human plasma was used to treat serum-starved HPAECs (1% v / v final concentration), and BMP activity was measured by ID1 gene induction. All the ID1 -induction activity from 1% plasma can be completely inhibited by ALK1-Fc (at 2.5 μg/ml) alone, suggesting that all the ID1 -gene induction activity in 1% plasma was due to BMP9 and BMP10. While most of this activity can be inhibited by anti-BMP9 antibody (at 15 μg/ml), the residual ID1 -induction activity cannot be inhibited by additional amounts of anti-BMP9 antibody (at 20 μg/ml). It can be only inhibited by either anti-BMP10 antibody (at 15 μg/ml) or ALK1-Fc (at 2.5 μg/ml), suggesting the residual ID1 -induction activity is due to BMP10. n = 3, one-way ANOVA, Dunnett's post test. *, p ≤ 0.05; #, p ≤ 0.05; ##, p ≤ 0.01; n.s ., not significant.

Article Snippet: Anti-BMP9 antibody (MAB3209), anti-BMP10 antibody (MAB2926), BMP10 prodomain (3956-BP-050), anti-BMP10 propeptide antibody (AF3956), biotinylated anti-BMP10 propeptide antibody (BAF3956), ALK1-Fc (370-AL), human BMP10 GFD (2926-BP-025) were all purchased from R&D Systems, Inc. Anti-phosphoSmad1/5/8 and anti-phosphoSmad1/5 antibody were purchased from Cell Signaling Technology.

Techniques: Derivative Assay, Clinical Proteomics, Expressing, Activity Assay, Cell Culture, Gene Expression, Control, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: The Prodomain-bound Form of Bone Morphogenetic Protein 10 Is Biologically Active on Endothelial Cells *

doi: 10.1074/jbc.M115.683292

Figure Lengend Snippet: Right-atrium-secreted active BMP10 is in the prodomain-bound form. Proteins from RA-conditioned medium were separated by gel filtration chromatography, and the BMP activities in the fractions were measured using the BRE-luciferase assay as described in “Experimental Procedures.” The relative BRE-luciferase activities in the fractions were plotted against their elution volumes ( black line with circle symbols ). The same process was repeated with diluted, purified recombinant human pBMP10, and the relative BMP activities in the fractions were also measured and plotted ( gray line with square symbols ). In addition, a pBMP10-specific ELISA was carried out to measure the proteins in the fractions from RA-conditioned medium gel filtration. Data were presented as optical densities (absorbance at 405 nm, right axis, dotted line with triangular symbols ). Proteins from Gel Filtration Calibration Kit were run under identical conditions, and arrows below showed the elution volumes of the protein standards.

Article Snippet: Anti-BMP9 antibody (MAB3209), anti-BMP10 antibody (MAB2926), BMP10 prodomain (3956-BP-050), anti-BMP10 propeptide antibody (AF3956), biotinylated anti-BMP10 propeptide antibody (BAF3956), ALK1-Fc (370-AL), human BMP10 GFD (2926-BP-025) were all purchased from R&D Systems, Inc. Anti-phosphoSmad1/5/8 and anti-phosphoSmad1/5 antibody were purchased from Cell Signaling Technology.

Techniques: Filtration, Chromatography, Luciferase, Purification, Recombinant, Enzyme-linked Immunosorbent Assay