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: Journal of Translational Medicine

Article Title: Bone morphogenetic protein 10 serves as a biomarker and a potential therapeutic target for endothelial dysfunction in endotoxin-induced acute lung injury

doi: 10.1186/s12967-025-06742-6

Figure Lengend Snippet: BMP10 ameliorates LPS-induced acute lung injury and inflammation. A H&E staining of lung sections demonstrated that BMP10 treatment mitigated LPS-induced thickening of the alveolar septal walls, reduced infiltration of inflammatory cells within the interstitium, and preserved the alveolar structure; Scale bar, 100 μm. B LPS-stimulated mice treated with BMP10 exhibited a significantly lower lung injury score compared with the LPS group ( *p < 0.05, n = 4 mice per group); Data were presented as mean ± standard error of mean, and groups were analyzed by two-tailed non-parametric test (Mann–Whitney U test). C IF staining of BALF indicated that BMP10 treatment significantly decreased the recruitment of activated neutrophils into the alveolar space induced by LPS ( *p < 0.05, n = 4 mice per group); Data were presented as mean ± standard error of mean, and groups were analyzed by two-tailed non-parametric test (Mann–Whitney U test); Scale bar, 50 µm; Red, MPO; Green, Ly6G; Blue, DAPI; D The levels of proinflammatory cytokines, including TNFα and IL-6, in the BALF, blood, and pulmonary homogenate supernatants were significantly lower in BMP10-treated, LPS-stimulated mice compared with the LPS-stimulated mice group ( *p < 0.05, n = 3—5 mice per group); Data were presented as mean ± standard error of mean, and groups were analyzed by two-tailed non-parametric test (Mann–Whitney U test). BMP10 bone morphogenetic protein 10, H&E hematoxylin and eosin, LPS lipopolysaccharide, IF immunofluorescence, BALF bronchoalveolar lavage fluid, TNF-α tumor necrosis factor alpha, MPO myeloperoxidase, Ly6G lymphocyte antigen 6 complex locus G6D

Article Snippet: After a 2-h interval, mice were treated with recombinant mouse BMP10 (6038-BP-025/CF, R&D system, Minneapolis, MN, USA) with a single dose of 1.0 μg i.p.

Techniques: Staining, Two Tailed Test, MANN-WHITNEY, Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: Bone morphogenetic protein 10 serves as a biomarker and a potential therapeutic target for endothelial dysfunction in endotoxin-induced acute lung injury

doi: 10.1186/s12967-025-06742-6

Figure Lengend Snippet: BMP10 mitigated LPS-induced increasing murine pulmonary endothelial permeability. A TEM of murine lung sections revealed that BMP10 treatment improved the LPS-induced disruption of pulmonary endothelial integrity and continuity, as well as reduced interstitial edema; Scale bar, 5 μm. B Quantitative IHC analysis of pulmonary VE-cadherin expression demonstrated that BMP10 treatment significantly inhibited the LPS-induced downregulation of VE-cadherin expression ( *p < 0.05, n = 3 mice per group); Scale bar, 100 μm; Data were presented as mean ± standard error of mean, and groups were analyzed by two-tailed non-parametric test (Mann–Whitney U test). C The total protein levels in the BALF were significantly lower in BMP10-treated, LPS-stimulated mice than in LPS-stimulated mice ( *p < 0.05, n = 4 mice per group); Data were presented as mean ± standard error of mean, and groups were analyzed by two-tailed non-parametric test (Mann–Whitney U test). TEM transmission electron microscopy, BMP10 bone morphogenetic protein 10, LPS lipopolysaccharide, IHC immunohistochemistry, BALF bronchoalveolar lavage fluid, VE-cadherin vascular endothelial cadherin

Article Snippet: After a 2-h interval, mice were treated with recombinant mouse BMP10 (6038-BP-025/CF, R&D system, Minneapolis, MN, USA) with a single dose of 1.0 μg i.p.

Techniques: Permeability, Disruption, Expressing, Two Tailed Test, MANN-WHITNEY, Transmission Assay, Electron Microscopy, Immunohistochemistry

Journal: Journal of Translational Medicine

Article Title: Bone morphogenetic protein 10 serves as a biomarker and a potential therapeutic target for endothelial dysfunction in endotoxin-induced acute lung injury

doi: 10.1186/s12967-025-06742-6

Figure Lengend Snippet: BMP10 inhibited LPS-induced murine pulmonary endothelial dysfunction and apoptosis. A Western blot analysis of murine lung homogenates revealed that VE-cadherin expression decreased, whereas the expression of angiopoietin-2, ICAM-1, and VCAM-1 increased following LPS stimulation. Treatment with BMP10 reversed these changes ( *p < 0.05, n = 4 mouse per group); Data were presented as mean ± standard error of mean, and groups were analyzed by two-tailed non-parametric test (Mann–Whitney U test). B IF staining of murine lung sections indicated that BMP10 treatment prevented the LPS-induced downregulation of MCL-1 expression; Scale bars, 100 µm; Green, MCL-1; Blue, DAPI. C TUNEL staining of murine lung sections demonstrated that BMP10 treatment effectively inhibited LPS-induced pulmonary apoptosis; Scale bar, 50 µm. BMP10 bone morphogenetic protein 10, IF immunofluorescence, VE-cadherin vascular endothelial cadherin, ICAM-1 intercellular adhesion molecule 1, VCAM-1 vascular cell adhesion protein 1, LPS lipopolysaccharide, MCL-1 myeloid cell leukemia sequence 1, BCL-2 B-cell leukemia/lymphoma type 2, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling

Article Snippet: After a 2-h interval, mice were treated with recombinant mouse BMP10 (6038-BP-025/CF, R&D system, Minneapolis, MN, USA) with a single dose of 1.0 μg i.p.

Techniques: Western Blot, Expressing, Two Tailed Test, MANN-WHITNEY, Staining, TUNEL Assay, Immunofluorescence, Sequencing

Journal: Journal of Translational Medicine

Article Title: Bone morphogenetic protein 10 serves as a biomarker and a potential therapeutic target for endothelial dysfunction in endotoxin-induced acute lung injury

doi: 10.1186/s12967-025-06742-6

Figure Lengend Snippet: BMP10 alleviated LPS-induced endothelial dysfunction both in vitro and in vivo through the canonical signaling pathway. HPMECs were cultured with 100 ng/ml of BMP10 for 24 h, followed by exposure to 10 μg/ml of LPS for a predetermined duration based on the study design. A Western blot analysis showed that 24 h of LPS stimulation significantly increased the protein expression levels of ICAM-1 and VCAM-1 in HPMECs. However, these changes were reversed by BMP10 treatment ( *p < 0.05, n = 4 per group); Data are presented as mean ± standard error of the mean, and group comparisons were analyzed using a two-tailed non-parametric test (Mann–Whitney U test). B IF staining of HPMECs demonstrated that BMP10 prevented the LPS-induced reduction in the expression of VE-cadherin and pSmad1/5/8, a marker of the BMP10-activated canonical signaling pathway, following 2 h of LPS stimulation; scale bars, 100 µm; Green, VE-cadherin; Red, pSmad1/5/8; Blue, DAPI. C Western blot analysis of lung homogenates revealed that 24 h of LPS stimulation significantly increased pSmad1/5/8 protein levels, but BMP10 treatment reversed these effects ( *p < 0.05, n = 4 per group); Data are presented as mean ± standard error of the mean, and group comparisons were analyzed using a two-tailed non-parametric test (Mann–Whitney U test). D Western blot analysis of HPMECs showed that 6 h of LPS stimulation significantly increased pSmad1/5/8 protein expression, which was similarly reversed by BMP10 pretreatment ( *p < 0.05, n = 4 per group); Data are presented as mean ± standard error of the mean, and groups were analyzed using a two-tailed non-parametric test (Mann–Whitney U test). HPMEC human pulmonary microvascular endothelial cell, LPS lipopolysaccharide, BMP10 bone morphogenetic protein 10, ICAM-1 intercellular adhesion molecule 1, VCAM-1 vascular cell adhesion protein 1, VE-cadherin vascular endothelial cadherin, pSmad1/5/8 phosphorylated small mother against decapentaplegic 1/5/8

Article Snippet: After a 2-h interval, mice were treated with recombinant mouse BMP10 (6038-BP-025/CF, R&D system, Minneapolis, MN, USA) with a single dose of 1.0 μg i.p.

Techniques: In Vitro, In Vivo, Cell Culture, Western Blot, Expressing, Two Tailed Test, MANN-WHITNEY, Staining, Marker

Journal: Journal of Translational Medicine

Article Title: Bone morphogenetic protein 10 serves as a biomarker and a potential therapeutic target for endothelial dysfunction in endotoxin-induced acute lung injury

doi: 10.1186/s12967-025-06742-6

Figure Lengend Snippet: BMP10 inhibited LPS-induced in vitro human pulmonary endothelial apoptosis. A TUNEL staining showed that BMP10 treatment effectively suppressed apoptosis of HPMECs induced by 24 h of LPS stimulation; scale bars, 100 µm. B IF staining of HPMECs demonstrated that BMP10 treatment inhibited the downregulation of MCL-1 expression caused by 24 h of LPS incubation; scale bars, 100 µm; Green, MCL-1; Blue, DAPI. C Western blot analysis of HPMECs showed an elevation in cleaved caspase 3 protein levels after 6 h of LPS stimulation, and treatment with BMP10 effectively inhibited caspase 3 cleavage ( *p < 0.05, n = 4 mouse per group); Data were presented as mean ± standard error of mean, and groups were analyzed by two-tailed non-parametric test (Mann–Whitney U test). TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, BMP10 bone morphogenetic protein 10, LPS lipopolysaccharide, IF immunofluorescence, HPMEC human pulmonary microvascular endovascular cell, MCL-1 myeloid cell leukemia sequence 1

Article Snippet: After a 2-h interval, mice were treated with recombinant mouse BMP10 (6038-BP-025/CF, R&D system, Minneapolis, MN, USA) with a single dose of 1.0 μg i.p.

Techniques: In Vitro, TUNEL Assay, Staining, Expressing, Incubation, Western Blot, Two Tailed Test, MANN-WHITNEY, Immunofluorescence, Sequencing

Journal: Journal of Translational Medicine

Article Title: Bone morphogenetic protein 10 serves as a biomarker and a potential therapeutic target for endothelial dysfunction in endotoxin-induced acute lung injury

doi: 10.1186/s12967-025-06742-6

Figure Lengend Snippet: BMP10 is a biomarker for predicting mortality in ICU patients diagnosed with pneumonia-related acute respiratory failure requiring invasive mechanical ventilation. A Plasma levels of BMP10 on the day of recruitment and B on day 2 after recruitment were significantly higher in patients who died in the hospital than in those who survived; Data were presented as medians with interquartile ranges (IQR) and groups were analyzed by Mann–Whitney U test; BMP10 bone morphogenetic protein 10

Article Snippet: After a 2-h interval, mice were treated with recombinant mouse BMP10 (6038-BP-025/CF, R&D system, Minneapolis, MN, USA) with a single dose of 1.0 μg i.p.

Techniques: Biomarker Discovery, Clinical Proteomics, MANN-WHITNEY

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