Journal: Cell communication and signaling : CCS

Article Title: FKBP12 is a major regulator of ALK2 activity in multiple myeloma cells.

doi: 10.1186/s12964-022-01033-9

Figure Lengend Snippet: Fig. 5 Proposed regulation of ligand dependent ALK2 activity in multiple myeloma cells. A ALK2 ligands such as BMP6, BMP9, and activin B signal via ALK2 preferably in complex with ACVR2A or ACVR2B. Activin A also binds to ALK2:ACVR2 but may either form a non-signaling complex (NSC) or an active signaling complex depending on the context. Addition of FK506 removes FKBP12 from the activation domain of ALK2 leading to increased ligand-induced SMAD1/5-activation. SMAD1/5-activation leads to myeloma cell apoptosis. B ALK3 ligands such as BMP2, BMP4, and BMP10, do not activate SMAD1/5 via ALK2 even in the presence of their preferred type II receptor BMPR2. It is unclear if the ligand can form an NSC with the receptors or not. Addition of FK506 removes FKBP12 from the activation domain of ALK2 leading to a dose-dependent ligand-induced SMAD1/5-activation and myeloma cell apoptosis. The figure was made with Biorender.com

Article Snippet: Recombinant human BMP2 (#355-BM), BMP4 (#314-BP), BMP6 (#507-BP), BMP9 (#3209-BP), BMP10 (#2926-BP), TGF-β1 (#240-B), and activin B (#659-AB-025) were from R&D Systems (BioTechne, Abingdon, UK).

Techniques: Activity Assay, Activation Assay

Journal: Angiogenesis

Article Title: BMP10 functions independently from BMP9 for the development of a proper arteriovenous network.

doi: 10.1007/s10456-022-09859-0

Figure Lengend Snippet: Fig. 8 BMP10, but not BMP9, suppresses the development of AVMs caused by ENG- deficiency. a–e CD31 and SMA immunofluorescence staining on retinas isolated from P7 control (PBS-treated Scl-CreER-negative Eng-iKO, a, n = 28 retinas), PBS-treated Scl-CreER;Eng-iKO (b, n = 16), BMP9-treated Scl-CreER;Eng- iKO (c, n = 8), BMP10-treated Scl-CreER;Eng-iKO (d, n = 8), and BMP9/BMP10-treated Scl-CreER;Eng-iKO (e, n = 10) mice. Arrows mark arterio- venous shunts. a artery, v vein. Scale bars, 500 μm. f Quantifi- cation of the number of AVMs. Data are mean ± SD. One-way ANOVA followed by Tukey’s post hoc test

Article Snippet: Control PBS, 100 ng of mouse BMP9 protein (R&D systems, 5566-BP), or 100 ng of mouse BMP10 protein (R&D systems, 6038-BP) was injected intraperitoneally and daily on the opposite side of the milk spot until the sample collection.

Techniques: Immunofluorescence, Staining, Isolation, Control

Journal: Journal of cellular physiology

Article Title: Combined Effects of Bone Morphogenetic Protein 10 and Crossveinless-2 on Cardiomyocyte Differentiation in Mouse Adipocyte-Derived Stem Cells

doi: 10.1002/jcp.25983

Figure Lengend Snippet: Mouse DFAT cells were treated with growth medium supplemented with control vehicle, BMP10 (25 ng/ml), CV2 (50 ng/ml), or BMP10 for 3 days followed by CV2 for 3 days (BMP10/CV2). Photos were obtained after 5, 7, 10 and 14 days.

Article Snippet: There was no detectable BMP10 in the culture medium (20% FBS) when tested by BMP10 DuoSet ELISA (R&D Systems, mature bovine BMP10 is 100% identical to human BMP10).

Techniques: Control

Journal: Journal of cellular physiology

Article Title: Combined Effects of Bone Morphogenetic Protein 10 and Crossveinless-2 on Cardiomyocyte Differentiation in Mouse Adipocyte-Derived Stem Cells

doi: 10.1002/jcp.25983

Figure Lengend Snippet: Mouse DFAT cells were treated with growth medium supplemented with control vehicle (day 5-7), BMP10 (25 ng/ml, day 5-7), CV2/5-7 (50 ng/ml, day 5-7), BMP10 (day 5-7) followed by CV2 (day 8-10) (BMP10/CV2), anti-BMP10 antibodies (100 ng/ml, day 5-7), or CV2/8-10 (50 ng/ml, day 8-10).

Article Snippet: There was no detectable BMP10 in the culture medium (20% FBS) when tested by BMP10 DuoSet ELISA (R&D Systems, mature bovine BMP10 is 100% identical to human BMP10).

Techniques: Control

Journal: Journal of cellular physiology

Article Title: Combined Effects of Bone Morphogenetic Protein 10 and Crossveinless-2 on Cardiomyocyte Differentiation in Mouse Adipocyte-Derived Stem Cells

doi: 10.1002/jcp.25983

Figure Lengend Snippet: Mouse DFAT cells were treated with growth medium supplemented with control vehicle (day 5-7), BMP10 (25 ng/ml, day 5-7), CV2/5-7 (50 ng/ml, day 5-7), BMP10 (day 5-7) followed by CV2 (day 8-10) (BMP10/CV2). Cell morphology and expression of sarcomeric alpha-actinin (Sr-alpha-actinin, green) and Troponin I (Tr-I, red) were examined after 14 days by immunofluorescence. DAPI (blue) was used to visualize nuclei.

Article Snippet: There was no detectable BMP10 in the culture medium (20% FBS) when tested by BMP10 DuoSet ELISA (R&D Systems, mature bovine BMP10 is 100% identical to human BMP10).

Techniques: Control, Expressing, Immunofluorescence

Journal: Journal of cellular physiology

Article Title: Combined Effects of Bone Morphogenetic Protein 10 and Crossveinless-2 on Cardiomyocyte Differentiation in Mouse Adipocyte-Derived Stem Cells

doi: 10.1002/jcp.25983

Figure Lengend Snippet: Mouse DFAT cells were treated with growth medium supplemented with control vehicle (day 5-7), BMP10 (25 ng/ml, day 5-7), CV2/5-7 (50 ng/ml, day 5-7), BMP10 (day 5-7) followed by CV2 (day 8-10) (BMP10/CV2). Cell morphology and expression of sarcomeric alpha-actinin (Sr-alpha-actinin, green) and Troponin I (Tr-I, red) were examined after 3 weeks by immunofluorescence. DAPI (blue) was used to visualize nuclei.

Article Snippet: There was no detectable BMP10 in the culture medium (20% FBS) when tested by BMP10 DuoSet ELISA (R&D Systems, mature bovine BMP10 is 100% identical to human BMP10).

Techniques: Control, Expressing, Immunofluorescence

Journal: Journal of cellular physiology

Article Title: Combined Effects of Bone Morphogenetic Protein 10 and Crossveinless-2 on Cardiomyocyte Differentiation in Mouse Adipocyte-Derived Stem Cells

doi: 10.1002/jcp.25983

Figure Lengend Snippet: (A, B) Mouse DFAT cells were plated in 6-well culture dishes and treated with growth medium supplemented with control vehicle, BMP10 (25 ng/ml), CV2 (50 ng/ml), or BMP10 for 3 days followed by CV2 for 3 days (BMP10/CV2). After 3 weeks, the cells were immunostained for Troponin I. For each treatment, all stained cells and colonies in one entire 6-well were counted by fluorescence microscopy. (A) The cells were classified as small round cells (<50 μm), or short (50-200 μm), medium (200-500 μm), or long myotubes (>500 μm), and (B) colonies were classified as small (1-25 myotubes), medium (25-50 myotubes) or large (>50 myotubes) colonies (representative of 3 experiments).

Article Snippet: There was no detectable BMP10 in the culture medium (20% FBS) when tested by BMP10 DuoSet ELISA (R&D Systems, mature bovine BMP10 is 100% identical to human BMP10).

Techniques: Control, Staining, Fluorescence, Microscopy

Journal: Stem Cell Reports

Article Title: Cardiosphere-Derived Cells Require Endoglin for Paracrine-Mediated Angiogenesis

doi: 10.1016/j.stemcr.2017.04.015

Figure Lengend Snippet: Endoglin-Deficient CDCs Show Reduced Endogenous SMAD1/5/8 Activation that Can Be Rescued by BMP9 Treatment (A) Summary of TGFβ and BMP9 signaling pathways. TGFβ/ALK5 and BMP9/ALK1 signaling are propagated through phosphorylation of SMAD2/3 and SMAD1/5/8, respectively. TGFβ generally signals via a complex of TGFBR2 and ALK5 (upper left) while BMP9 (and BMP10) signals via a complex of BMPR2 and ALK1 (upper right). Endoglin acts as a co-receptor to promote BMP9/10 signaling through the ALK1 receptor complex, although it can also promote TGFβ signaling through ALK1 to activate SMAD1/5/8 (not shown). (B and C) Representative western blots show that baseline and TGFβ1-induced SMAD2 phosphorylation (B) and SMAD3 phosphorylation (C) are similar in control and in Eng KO CDCs. Densitometric analysis of pSMAD2 and pSMAD3 band intensity relative to α-tubulin are shown as mean ± SEM from three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01. (D) Representative western blot shows SMAD1/5/8 phosphorylation is reduced in Eng KO CDCs and this is partially rescued following treatment with 2 ng/mL BMP9 for 30 min. Densitometric analysis of pSMAD1/5/8 band intensity relative to α-tubulin is shown as mean ± SEM from five independent experiments; ∗ p < 0.05.

Article Snippet: Commercial Taqman probes for Eng (Mm00468252_m1), Bmp9 (Mm00807340_m1), Bmp10 (Mm01183889_m1), Tgfbeta1 (Mm01178820_m1), Tgfbeta3 (Mm00436960_m1), Gapdh (Mm99999915_g1), and Hprt1 (Mm00446968_m1) were from Life Technologies and used for qPCR on a Quantstudio 6&7 Flex Real-Time PCR system.

Techniques: Activation Assay, Protein-Protein interactions, Phospho-proteomics, Western Blot, Control

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