reference mouse anti bmp9 antibody  (R&D Systems)

Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: An angiopoietin-2 vaccine improves arteriovenous malformation pathology in hereditary hemorrhagic telangiectasia mice

doi: 10.1016/j.bvth.2026.100155

Figure Lengend Snippet: Immunogenicity of the ANG2-P3:CRM197 vaccine in C57BL/6 mice. (A) Schematic diagram of the vaccination schedule and generation of the BMP9/10ib model (created with biorender.com ). (B) Protein sequence alignment of the C-terminal end of human ANG2 (hANG2), mouse ANG2 (mANG2), human ANG1 (hANG1), and mouse ANG1 (mANG1), along with the peptide sequences of ANG2-P3 and ANG1-P3. (C-D) Serum antibody titers against ANG2-P3 (C) and ANG1-P3 (D) in ANG2-P3:CRM197-vaccinated females (Vac-1 to Vac-5) and controls (injected with saline [Sal-1 and Sal-2] or CRM197-only [CRM-1 to CRM-5]). The vaccinated females with the highest anti-ANG2-P3 titers were identified as “best responders” (marked with a red box). OD, optical density.

Article Snippet: After 3 additional washes with PBST, serial dilutions of individual mouse serum samples were prepared, along with a reference mouse anti-BMP9 antibody (no. MAB3209; R&D Systems) and a reference mouse anti-BMP10 antibody (no. MAB2926; R&D Systems), both diluted in 1% BSA in PBS.

Techniques: Immunopeptidomics, Sequencing, Injection, Saline

Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: An angiopoietin-2 vaccine improves arteriovenous malformation pathology in hereditary hemorrhagic telangiectasia mice

doi: 10.1016/j.bvth.2026.100155

Figure Lengend Snippet: ANG2-P3:CRM197 vaccine reduces AVM pathology in BMP9/10ib neonates. (A) Serum antibody titers against ANG2-P3 in pups and their corresponding dams vaccinated with ANG2-P3:CRM197 (Vac-1 to Vac-3) or injected with saline (Saline). Vac-1, Vac-2, and Vac-3 pups: n = 7; Saline pups: n = 2. Dams: n = 1. (B) Representative immunofluorescence staining with isolectin B4 (IB4; green) and of SMA (red) in P6 retinas of pups treated with PBS or BMP9/10ib from a dam vaccinated with ANG2-P3:CRM197 or injected with saline. Scale bar: 1.5 mm. (C-D ) AVM count per retina (C), and retinal AVM surface area (D) in BMP9/10ib pups from dams vaccinated with ANG2-P3:CRM197 or injected with saline (Saline). BMP9/10ib + saline: n = 24; BMP9/10ib + ANG2-P3:CRM197: n = 37 (C). BMP9/10ib + saline: n = 24; BMP9/10ib + ANG2-P3:CRM197: n = 34 (D). (E) Spearman rank correlation matrix of the indicated variables (n = 15). (F-H) Retinal artery diameter (F), retinal vein diameter (G), and SMA coverage area (H) in BMP9/10ib pups from dams vaccinated with ANG2-P3:CRM197 or injected with saline (Saline). Data are shown as mean ± standard error of the mean (SEM); unpaired t test with Welch correction for panel C, Mann-Whitney test for panel D, and 1-way analysis of variance with Tukey multiple comparisons test for panels F-H. PBS + saline: n = 15; BMP9/10ib + saline: n = 24; BMP9/10ib + ANG2-P3:CRM197: n = 37 (F,G). PBS + saline: n = 13; BMP9/10ib + saline: n = 21; BMP9/10ib + ANG2-P3:CRM197: n = 29 (H). ∗ P < .05; ∗∗∗ P ≤ .001; ∗∗∗∗ P < .0001. (I) Immunoassay data measuring ANG2 levels in plasma from patients with HHT and healthy controls. Data are shown as mean ± SEM; unpaired t test with Welch correction. Healthy: n = 36; HHT: n = 36. ∗∗ P ≤ .01. a, artery; ns, not significant; v, vein.

Article Snippet: After 3 additional washes with PBST, serial dilutions of individual mouse serum samples were prepared, along with a reference mouse anti-BMP9 antibody (no. MAB3209; R&D Systems) and a reference mouse anti-BMP10 antibody (no. MAB2926; R&D Systems), both diluted in 1% BSA in PBS.

Techniques: Injection, Saline, Immunofluorescence, Staining, MANN-WHITNEY, Clinical Proteomics

Journal: bioRxiv

Article Title: Physiological perfusion of human vasculature reveals a YAP/TAZ-Apelin switch linking intraluminal flow to endothelial state transitions and vessel remodeling

doi: 10.64898/2026.03.21.713033

Figure Lengend Snippet: (A) Vascular beds on Day 4 which were grown from ENG - or ALK1 -knockdown endothelial cells or treated with 10 ng/mL BMP9 (ERG: endothelial nuclei, CD31: vessels). (B) Quantification of (A) (10 devices per condition across 4 independent experiments for shRNA vessels; 6 devices per condition across 2 independent experiments for BMP9 treatments). (C) Particle image velocimetry analysis showing velocity magnitude for ENG -knockdown, ALK1 -knockdown, and BMP9-treated vessels on Day 4. (D) Quantification of (C) (42-45 vessels and 6 devices per condition across 2 independent experiments). (E) Gene set scoring of endothelial cells on Day 4 from the scRNA-seq experiment. Scores are for BMP9 up/down-regulated genes from Al Tabosh et al . . Endothelial cells are divided by either cell type (i.e. T1 or T2), predicted labels from Goveia et al . (i.e. tip cell or stalk-like), or experimental condition (i.e. low or high flow). Cohen’s d effect sizes are shown. (F) Correlation coefficients between gene set scoring of BMP9 up/down-regulated genes (Al Tabosh et al .), VEGFA up/down-regulated genes (Zhang et al ., ), and YAP/TAZ targets. Adjusted p-values were calculated using a permutation test followed by Bonferroni correction. (G) Gene set scoring of BMP9 down-regulated genes and VEGFA up-regulated genes. (H) Gene set scoring of BMP9 up-regulated genes and VEGFA down-regulated genes. (I) Log2 fold change rank plot showing a subset of markers genes for the T1 cell type (left). Gene set scoring for the subset of marker genes (right). (F-H) All endothelial cells from the scRNA-seq dataset were used. (J) Heatmap of qPCR gene expression from monolayer BMVECs treated with BMP9 (10 ng/mL) and/or VEGFA (100 ng/mL) for 48 hrs (3 independent experiments). (K) Images of ENG - or ALK1 -knockdown vascular beds on Day 4 treated with 100 nM Axitinib (CD31: vessels). (L) Quantification of (K) (6 devices per condition across 2 independent experiments). (B, D, L) 2-tailed unpaired Welch’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001). (B, L) Error bars show SEM.

Article Snippet: Human recombinant BMP9 (R&D Systems; 10 ng/mL) and/or human recombinant VEGF-165 (Gibco; 100 ng/mL) were added for 48 hrs.

Techniques: Knockdown, shRNA, Marker, Gene Expression

Journal: bioRxiv

Article Title: Physiological perfusion of human vasculature reveals a YAP/TAZ-Apelin switch linking intraluminal flow to endothelial state transitions and vessel remodeling

doi: 10.64898/2026.03.21.713033

Figure Lengend Snippet: (A) Evaluation of shRNA knockdown efficiency (2 independent experiments). (B) ERG and MKI67 immunostainings of vascular beds on Day 4 which were grown from shRNA-treated endothelial cells or additionally treated with BMP9 (10 ng/mL). (C) Quantifications of (B) (6 devices per condition across 2 independent experiments). 2-tailed unpaired student’s t-test (ns: not significant). (A, C) Error bars show SEM.

Article Snippet: Human recombinant BMP9 (R&D Systems; 10 ng/mL) and/or human recombinant VEGF-165 (Gibco; 100 ng/mL) were added for 48 hrs.

Techniques: shRNA, Knockdown

Journal: bioRxiv

Article Title: Physiological perfusion of human vasculature reveals a YAP/TAZ-Apelin switch linking intraluminal flow to endothelial state transitions and vessel remodeling

doi: 10.64898/2026.03.21.713033

Figure Lengend Snippet: (A) Gene Set Enrichment Analysis of VIVOS scRNA-seq data for BMP9 up/down-regulated genes (Al Tabosh et al. , ). (B) Left: Venn diagram between BMP9 down-regulated genes (Al Tabosh et al. ) and VEGFA up-regulated genes (Zhang et al. , ). Right: Gene set scoring of VIVOS scRNA-seq data for BMP9 down-regulated genes and VEGFA up-regulated genes, with overlapping genes removed. Endothelial cells from all conditions are shown. (C) Gene set scoring of the human tumor angiogenesis atlas from Goveia et al. for BMP9 down-regulated genes and VEGFA up-regulated genes. (D) Gene set scoring of VIVOS scRNA-seq data for BMP9 up/down-regulated genes and YAP/TAZ targets. T1 endothelial cells on Day 4 are shown. (E) Gene set scoring of VIVOS scRNA-seq data for BMP9 down-regulated genes and VEGFA up-regulated genes. Endothelial cells from Day 1 are shown. RNA velocity streamlines (left). Individual gene expressions (right). (F) Rank plot of differentially expressed genes for tip cell and stalk-like human atlas clusters. (G) Heatmap of qPCR gene expression from monolayer BMVECs treated with BMP9 (10 ng/mL) and/or VEGFA (100 ng/mL) for 48 hrs. Numbers represent average relative expressions from 3 independent experiments. “Mean expression” refers to the geometric mean of the 7 individual genes. (H) Mean expression values from (G). (I) VEGFR1 and VEGFR2 expressions from the same experiment as (G). (J) VEGFR1 expression in endothelial cells from all conditions. (K) Proposed model for antagonization of VEGFA-induced transcription by BMP9. (B-E) Spearman’s correlation coefficient. (H-I) 2-tailed unpaired student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001). Error bars show SEM.

Article Snippet: Human recombinant BMP9 (R&D Systems; 10 ng/mL) and/or human recombinant VEGF-165 (Gibco; 100 ng/mL) were added for 48 hrs.

Techniques: Gene Expression, Expressing

Journal: Biomedicines

Article Title: Recombinant BMP9 Reinforces Gut Vascular Barrier in Experimental Colitis

doi: 10.3390/biomedicines14020288

Figure Lengend Snippet: Dysregulation of BMP9/ALK1 signaling and inflammation in refractory ulcerative colitis (rUC). ( A ) Heatmap depicting serum expression levels of BMP family members in healthy controls, non-rUC, and rUC patients ( n = 3 per group). Data were Z-score normalized and hierarchically clustered (red, high expression; blue, low expression). ( B ) Baseline serum levels of BMP9 and BMP10 in healthy controls ( n = 50), non-rUC patients ( n = 47), and rUC patients ( n = 48). ( C ) Spearman correlation analyses between baseline serum BMP9 levels and clinical disease activity indices, including baseline Modified Mayo Score, baseline UCEIS, post-treatment Modified Mayo Score, and post-treatment UCEIS, in patients with UC ( n = 95). ( D ) Colonic mucosal mRNA expression levels of ALK1, IL-6, TNF-α, and CCL2 in healthy controls, non-rUC, and rUC patients ( n = 4 per group). Statistical annotations for ( B , D ): (Normalized to GAPDH; Mean ± SD; Statistical significance determined by one-way ANOVA with Tukey’s post hoc test: ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: Recombinant murine BMP9 (MCE, HY-P700530) was administered via intraperitoneal injection (200 ng/mouse/day) from day 0 to day 7, concurrent with DSS induction.

Techniques: Expressing, Activity Assay, Modification

Journal: Biomedicines

Article Title: Recombinant BMP9 Reinforces Gut Vascular Barrier in Experimental Colitis

doi: 10.3390/biomedicines14020288

Figure Lengend Snippet: BMP9 attenuates DSS-induced colitis in mice. ( A ) Schematic of experimental design: Acute colitis was induced in C57BL/6 mice by 3% DSS in drinking water for 7 days. The BMP9 treatment group received intraperitoneal injections of recombinant murine BMP9 (200 ng/day), while the DSS group and the control group received PBS ( n = 14 per group). ( B ) Serum BMP9(ng/mL) concentrations measured by ELISA. ( C ) Representative images of colons and quantitative analysis of colon length (cm). ( D ) Colonoscopy images (upper), H&E-stained colon sections (lower; scale bars = 100 μm), and histopathological scores. ( E ) (Upper) Dynamic body weight changes and (Lower) Disease Activity Index (DAI) scores. Data expressed as mean ± SD; * p < 0.05, two-way repeated measures ANOVA. ( F ) (Left) Relative Alk1 mRNA expression in colon tissues (RT-qPCR normalized to Gapdh). (Right) ALK1 protein concentrations (quantified by ELISA). Data presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 ns: not significant). ( G ) Relative mRNA expression levels of IL-1β, Ccl2, Col1a1, and Col3a1 (RT-qPCR; mean ± SD). ( H ) Western blot analysis of CCL2, TGF-β, and α-SMA protein expression in colon tissues and quantitative analysis of band intensities. ( I ) Western blot detection of p-Smad1, total Smad1, and VE-cadherin proteins and quantitative analysis of band intensities.

Article Snippet: Recombinant murine BMP9 (MCE, HY-P700530) was administered via intraperitoneal injection (200 ng/mouse/day) from day 0 to day 7, concurrent with DSS induction.

Techniques: Recombinant, Control, Enzyme-linked Immunosorbent Assay, Staining, Activity Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Biomedicines

Article Title: Recombinant BMP9 Reinforces Gut Vascular Barrier in Experimental Colitis

doi: 10.3390/biomedicines14020288

Figure Lengend Snippet: BMP9 restores intestinal vascular barrier integrity in DSS-induced colitis. ( A ) Representative immunofluorescence images of VE-cadherin (red) and CD31 (green) co-localization in colon tissues (nuclei counterstained with DAPI). ( B ) Left: Schematic of FITC-dextran (4 kDa) permeability assay. Right: Quantified serum FITC fluorescence intensity 60 min post-gavage ( n = 5 per group). ( C ) Left: Schematic of Evans Blue vascular leakage assay. Right: Colonic Evans Blue extravasation quantified by absorbance at 620 nm (mean ± SD; n = 5 per group; ** p < 0.01, *** p < 0.001, ns: not significant; one-way ANOVA with Tukey’s test). ( D ) KEGG pathway analysis of RNA-seq data from colon tissues (DSS + BMP9 groups vs. DSS). ( E ) Volcano plot of differentially expressed genes. Genes highlighted in bold are key IBD-associated downregulated factors.

Article Snippet: Recombinant murine BMP9 (MCE, HY-P700530) was administered via intraperitoneal injection (200 ng/mouse/day) from day 0 to day 7, concurrent with DSS induction.

Techniques: Immunofluorescence, Permeability, Fluorescence, RNA Sequencing

Journal: Biomedicines

Article Title: Recombinant BMP9 Reinforces Gut Vascular Barrier in Experimental Colitis

doi: 10.3390/biomedicines14020288

Figure Lengend Snippet: BMP9/ALK1 signaling modulates neutrophil migration and endothelial tube formation. ( A ) Schematic representation of the neutrophil migration assay. ( B ) (Left) Representative fluorescence micrographs demonstrating Calcein-AM-labeled neutrophil migration through 3 μm pore Transwell inserts toward HIMECs pretreated for 2 h with: vehicle control (0 ng/mL BMP9), BMP9 (0.1, 1, or 10 ng/mL), TNF-α (20 ng/mL as positive control), or ALK1 inhibitor ML347 (150 nM). (Right) Quantitative analysis of neutrophil migration rates (mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant by two-way ANOVA with post hoc testing). ( C ) Schematic illustration of the endothelial tube formation assay protocol. ( D ) Representative phase-contrast images of tubular network formation by HIMECs cultured on growth factor-reduced Matrigel under various treatment conditions: vehicle control (0 ng/mL BMP9), BMP9 (0.1, 1, or 10 ng/mL), TNF-α (20 ng/mL), or ML347 (150 nM). ( E ) Quantitative assessment of angiogenic parameters including branch points and nodal junctions (mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 by two-way ANOVA with appropriate post hoc comparisons).

Article Snippet: Recombinant murine BMP9 (MCE, HY-P700530) was administered via intraperitoneal injection (200 ng/mouse/day) from day 0 to day 7, concurrent with DSS induction.

Techniques: Migration, Fluorescence, Labeling, Control, Positive Control, Endothelial Tube Formation Assay, Cell Culture

Journal: bioRxiv

Article Title: BMP9-mediated regulation of endothelin-1 requires integrated SMAD1/5 and SMAD2/3 signaling

doi: 10.64898/2025.12.24.696445

Figure Lengend Snippet: (A) UMAP plots depict distinct cell clusters in control (Ctrl) versus PAH explanted lungs identified by single cell RNAseq. EDN1 expression is highly enriched in the endothelial cluster of cells in both control and PAH lungs, and is increased in the lungs of PAH versus control lungs (GSE169471). (B) Volcano plot depicts upregulated and downregulated genes in explanted lungs from patients with PAH versus non-diseased controls. EDN1 is one of the most highly and significantly upregulated genes (GSE169471). (C) ET-1 levels were measured in plasma from normoxic (Nx) adult rats, and rats injected with SU5416 (40 mg/kg s.c.) and exposed to 3 weeks of hypoxia (FIO2=0.1) followed by 3 weeks of normoxia (SU-Hx). (D-E) Bovine aortic ECs (BAECs) were deprived of serum for 16 h and treated with varying concentrations of BMP9 (0.1, 1, 10 ng/mL) or TGFβ1 (5 ng/mL), and supernatants analyzed at (D) 6 h or (E) 24 h for presence of ET-1 peptide by ELISA, revealing dose-dependent effects. (F) Analysis of Edn1 mRNA by qRT-PCR in BAECs stimulated with BMP9 (25 ng/mL) revealed time-dependent effects. (G) Immunoblotting of BAECs stimulated with varying concentrations (0-50 ng/mL) of BMP9 for 25 min demonstrated activation of SMADs 1/5/9 in a dose-dependent manner. (H) Immunoblotting of BAECs stimulated with BMP9 or TGFβ1 revealed kinetics of SMAD2 and SMAD3 activation. Values are mean ± SEM, n=5, * p ≤0.05 and ** p <0.01 as compared to basal transcriptional levels, 1-Way ANOVA, Kruskal-Wallis Test.

Article Snippet: Recombinant ALK1 extracellular domain expressed as a soluble fusion protein with IgG Fc domain (ALK1-Fc), a neutralizing anti-BMP9 monoclonal antibody (MAB3209), and recombinant BMP inhibitor, Noggin, were obtained from R&D Systems.

Techniques: Control, Expressing, Clinical Proteomics, Injection, Peptide ELISA, Quantitative RT-PCR, Western Blot, Activation Assay

Journal: bioRxiv

Article Title: BMP9-mediated regulation of endothelin-1 requires integrated SMAD1/5 and SMAD2/3 signaling

doi: 10.64898/2025.12.24.696445

Figure Lengend Snippet: (A) Immunoblot of BAECs treated with BMP9 (5 ng/mL) revealed differential sensitivity of SMAD1 vs. SMAD3 activation ( p -SMAD1, p -SMAD3) to ALK1/2/3/6 kinase inhibition with varying concentrations of LDN-193189, vs. total SMAD1 ( t -SMAD1). (B) Immunoblot of matched ALK1 wild-type and knockout ( Acvrl1 flox/flox and Acvrl1 -/- ) mouse microvascular lung ECs (MLECs) treated with BMP9 (5 ng/mL) demonstrate absence of BMP9-mediated SMAD3 activation in ALK1 knockout cells, diminished SMAD1 activation, and more potent inhibition of SMAD1 activation by LDN-193189 in the absence of ALK1. (C) Immunoblot of BAECs stimulated with BMP9 (25 ng/mL ) or TGFβ1 (5 ng/mL) revealed high sensitivity of BMP9-mediated SMAD3 activation and partial sensitivity of SMAD1 activation in response to ALK1/2/3/6 inhibition (250 nM LDN-193189), and modest sensitivity of BMP9-mediated SMAD3 activation and no sensitivity of SMAD1 activation in response to ALK4/5/7 inhibition (5 μM SB-431542), while activation of SMAD3 or SMAD1 by TGFβ1 was sensitive to ALK4/5/7 inhibition. (D) Edn1 mRNA expression measured by RT-PCR in BAECs stimulated with BMP9 (25 ng/mL) or TGFβ1 (5 ng/mL) revealed differential sensitivity of BMP9- vs. TGFβ1-medated Edn1 expression to ALK1/2/3/6 (250 nM LDN-193189) or ALK4/57 inhibition (5 µM SB-431542) inhibition. (E) Treatment of BAECs with SMAD3 activation inhibitor SIS3 (5 μM) prevented both BMP9- and TGFβ1-induced expression of Edn1 mRNA by RT-CR. (F) BMP9-mediated expression of Edn1 mRNA in BAECs was inhibited by pretreatment with siRNA specific for SMAD3 but not SMAD1 by RT-PCR. (G) BMP9-induced expression of EDN1 mRNA in TIME cells was inhibited by pre-treatment with siRNA specific for SMAD2 or SMAD3, but not siRNA specific for SMAD1, SMAD5, or SMAD9. Bars represent mean ± SEM, n=3-4 as shown, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, 2-Way ANOVA with Holm-Sidak Test (D, G) or Fisher’s LSD test (E, F).

Article Snippet: Recombinant ALK1 extracellular domain expressed as a soluble fusion protein with IgG Fc domain (ALK1-Fc), a neutralizing anti-BMP9 monoclonal antibody (MAB3209), and recombinant BMP inhibitor, Noggin, were obtained from R&D Systems.

Techniques: Western Blot, Activation Assay, Inhibition, Knock-Out, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: BMP9-mediated regulation of endothelin-1 requires integrated SMAD1/5 and SMAD2/3 signaling

doi: 10.64898/2025.12.24.696445

Figure Lengend Snippet: (A-C) Immunoblot of HPAECs following siRNA-mediated knockdown of various BMP type I ( ALK1, ALK2, ALK3 ), type II receptors ( BMPR2, ACVR2A ) and co-receptors ( ENG ) revealed that ALK1 and BMPR2 are the primary mediators of SMAD3 activation in response to BMP9 (25 ng/mL), with little contribution from ALK2, ALK3, ALK5, ENG, or ACVR2A, and revealed ALK1 and the primary mediator of SMAD1 activation. (D) RT-PCR of HPAECs pre-treated with specific siRNA revealed inhibition of incremental BMP9-induced EDN1 expression by si ALK1 and si ENG , but only reduction of basal EDN1 expression by si ALK5 . (E) BMPR2 knockdown in HPAECs abrogated BMP9-mediated EDN1 expression by RT-PCR. (F) RT-PCR analysis of MLEC revealed loss of BMP9-induced (25 ng/mL) expression of Edn1 mRNA in Bmpr2 KO ( Bmpr2 -/- ) MLEC vs. wild-type MLECs ( Bmpr2 flox/flox ). (G) RT-PCR analysis of TIME cells pre-treated with specific siRNA revealed loss of BMP9-induced (1 ng/mL) expression of EDN1 mRNA following knockdown of BMPR2 and ALK1, but not ACVR2a or ALK2. (H-J) Pre-incubation of HPAECs with receptor extracellular domains fusion proteins (E) ALK1-Fc, (F) Eng-Fc, but not (G) ALK5-Fc inhibited BMP9-induced expression of EDN1 mRNA. Values are mean ± SEM, n=3-4, * p <0.05, ** p <0.01, **** p <0.0001, 2-Way ANOVA with Sidak’s Test.

Article Snippet: Recombinant ALK1 extracellular domain expressed as a soluble fusion protein with IgG Fc domain (ALK1-Fc), a neutralizing anti-BMP9 monoclonal antibody (MAB3209), and recombinant BMP inhibitor, Noggin, were obtained from R&D Systems.

Techniques: Western Blot, Knockdown, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Inhibition, Expressing, Incubation

Journal: bioRxiv

Article Title: BMP9-mediated regulation of endothelin-1 requires integrated SMAD1/5 and SMAD2/3 signaling

doi: 10.64898/2025.12.24.696445

Figure Lengend Snippet: EDN1 promoter activity induced by BMP9 versus TGFβ1 in BAECs was assayed using either wild type human EDN1 promoter of varying lengths or using a full-length promoter with mutations in BMP- and TGFβ-specific binding elements. (A) Previously described SMAD2/SMAD3 binding element (SBE1/SBE2), activator protein-1 (AP-1) binding site, and a putative NF-1 site with homology to a consensus SMAD1/5 binding element (“MEME2”) were required for EDN1 transcription in response to BMP9, whereas TGFβ only required the SBE1/SBE2 and AP-1 sites. (B-C) Mutant constructs were generated within a -650 bp version of the EDN1 promoter reporter construct and compared with the signaling of the wild-type construct by luciferase assay. Cells were deprived of fetal calf serum (FCS) for 16 h, and then treated with 25 ng/mL of BMP9 or 5 ng/mL of TGFβ1 for 12 h. Values are mean ± SEM, n=3-5, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, 2-Way ANOVA with Sidak’s Test, or in the case of promoter mutants, by Fisher’s exact test for each mutant / wild-type pair.

Article Snippet: Recombinant ALK1 extracellular domain expressed as a soluble fusion protein with IgG Fc domain (ALK1-Fc), a neutralizing anti-BMP9 monoclonal antibody (MAB3209), and recombinant BMP inhibitor, Noggin, were obtained from R&D Systems.

Techniques: Activity Assay, Binding Assay, Mutagenesis, Construct, Generated, Luciferase

Journal: bioRxiv

Article Title: BMP9-mediated regulation of endothelin-1 requires integrated SMAD1/5 and SMAD2/3 signaling

doi: 10.64898/2025.12.24.696445

Figure Lengend Snippet: The schematic illustrates the proposed mechanism demonstrated in this study. BMP9 initiates the formation of a membrane receptor complex consisting of two type I receptors (ALK1) with two type II receptors (BMPR2) and two ENG coreceptors, promoting transphosphorylation of the ALK1 kinase. Activated ALK1 then phosphorylates SMAD 2/3 and SMAD 1/5/9. Subsequently, pSMAD 2/3 and pSMAD 1/5/9 translocate to the nucleus where they regulate EDN1 transcription via binding to SBE1/SBE2 and NF1 promoter sites, respectively, in microvascular and macrovascular endothelial cells, thereby promoting PAH.

Article Snippet: Recombinant ALK1 extracellular domain expressed as a soluble fusion protein with IgG Fc domain (ALK1-Fc), a neutralizing anti-BMP9 monoclonal antibody (MAB3209), and recombinant BMP inhibitor, Noggin, were obtained from R&D Systems.

Techniques: Membrane, Binding Assay