Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in primary human foreskin fibroblasts (HFF-1) were determined by RT-qPCR. ( B ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated with IFNβ (5 ng/ml), BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), followed by either immunoblot analysis 1 h post stimulation or RNA extraction from cell lysates and RT-qPCR 6 h post stimulation. GAPDH transcript levels were used for normalization. ( C ) Immunoblot analysis of HFF-1 upon stimulation to determine phosphorylation levels of respective signaling components. ( D ) Transcript levels of the BMP-responsive genes Id1 and Id3 upon stimulation with the indicated ligands. ( E ) Transcript levels of the ISGs Isg15 , Irf9 , Ifi6 , and Stat2 upon stimulation with the indicated ligands. Data information: ( A , B ) The Experiment was performed two independent times, one representative is shown. ( D , E) The experiment was performed three independent times, one representative is shown. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Western Blot, RNA Extraction, Phospho-proteomics

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Presence of type I (ALK1, ALK2, ALK3) and type II (BMPR2) receptors in HFF-1 and 293T was verified by immunoblotting with the respective antibodies. Detection of GAPDH protein served as loading control. ( B ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in 293T were determined by RT-qPCR. ( C ) 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control (EF1α-Renilla). 24 h post transfection, 293T were either stimulated with BMP9 (3 nM), or BMP9 (3 nM) incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml or 5 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( D ) 293T were co-transfected as in ( B ). 24 h post transfection, 293 T were either stimulated with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or with the ligands incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( E ) HFF-1 were either mock, DMSO, Ruxolitinib (10 µM) or DMH1 (10 µM) treated for 2 h, followed by stimulation with IFNβ (5 ng/ml) or BMP4 (18 nM) for 2 h. Cells were lysed and lysates were subjected to immunoblot analysis with p-STAT1, STAT1, p-SMAD1/5/9, SMAD1, p-p38, p38, p-p44/42, p44/42, and Calnexin-specific antibodies. Data information: ( A – E ) Experiments were performed two independent times, one representative is shown. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples.

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Western Blot, Control, Quantitative RT-PCR, Transfection, Expressing, Luciferase, Incubation

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated for 6 h with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or co-stimulated with IFNβ (5 ng/ml), followed by HCMV infection (MOI 0.5) for 16 h. Cells were fixed, nuclei were stained and cells were labeled for HCMV IE1 + cells as a readout for infection. ( B ) HCMV IE1 + cells normalized to total cell numbers and the untreated control (white column) in BMP/Activin stimulated samples (left panel) or with IFNβ co-stimulated samples (right panel). ( C ) HCMV IE1 + cells normalized to total cell numbers in cells pre-stimulated with either low (0.25 nM) or high (3 nM) concentrations of BMP9 (green symbols) or IFNβ co-stimulated with low and high concentrations of BMP9 (beige symbols). ( D ) HFF-1 were infected by centrifugal enhancement with HCMV WT (MOI 0.5) and supernatants of infected cells were collected in 6 h increments. 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control. Twenty-four hours post transfection, 293T were either stimulated with supernatants from HCMV-infected cells, or supernatants from HCMV-infected cells incubated for 15 min at RT with an α-BMP9 antibody, for 16 h, followed by a dual-luciferase assay readout. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples. Data information: ( B ) Experiment was performed three independent times, one representative is shown. ( C , D ) Data are combined from two independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Infection, Staining, Labeling, Control, Transfection, Expressing, Luciferase, Incubation, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Schematic representation of the workflow. After 2 h of serum starvation, HFF-1 were stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR. ( B ) Transcript levels of BMP receptors Acvrl1 (ALK1, type I receptor) and Bmpr2 (type II receptor), which are the main receptors for BMP9, and the BMP-responsive gene Id1 . ( C ) Transcript levels of the ISGs Isg15 , Irf7 , Ifi6 , Irf9 , Stat2 , and Irf1 . ( D ) Transcript levels of the negative regulators Usp18 and Smurf1 . ( E ) HFF-1 were incubated with either Ruxolitinib or DMH1 for 1 h, then stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for transcript levels of Id3 , Stat2 , and Irf9 . Data information: ( B – D ) Data are combined from three independent experiments with the exception of Ifi6 transcript levels where three independent experiments were performed and data was combined from two independent experiments. ( E ) The experiment was performed two independent times, one representative is shown. Student’s t test (unpaired, two-tailed), n.s. not significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: RNA Extraction, Quantitative RT-PCR, Incubation, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) After 2 h of serum starvation, HFF-1 were stimulated for either one or 8 h with IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or were co-stimulated with IFNβ and BMP9, followed by cell lysis and immunoblot analysis with antibodies for phospho-STAT1, STAT1, phospho-SMAD1/5/9, and β-Actin. Phospho-STAT1 band intensities were first normalized to corresponding total STAT1 levels, then to IFNβ stimulation only, and are shown below. ( B ) After 2 h of serum starvation, HFF-1 were stimulated for 1 h with either IFNα2 (5 ng/ml), IFNγ (5 ng/ml), or BMP9 (3 nM) alone, or co-stimulated with IFNα2 or IFNγ and BMP9, followed by cell lysis, immunoblot analysis and quantification as in ( A ). ( C ) After 2 h of serum starvation, HFF-1 were stimulated for 6 h with either IFNα2 (5 ng/ml), IFNβ (5 ng/ml), IFNγ (5 ng/ml), or BMP9 (3 nM) alone, or co-stimulated with IFNα2, IFNβ, IFNγ, and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for Irf9 , Stat2 , Irf1 , and Id3 transcripts. Data information: ( A , B ) Experiment was performed three independent times, one representative immunoblot is shown. Quantified data for the STAT1 phosphorylation levels are combined from three independent experiments. ( C ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Lysis, Western Blot, RNA Extraction, Quantitative RT-PCR, Phospho-proteomics, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) 293T were co-transfected with expression plasmids for empty vector (EV) or V5-tagged US18, US20, or M27 (a known inhibitor of IFNAR signaling), together with either a BRE-Luciferase or MX1-Luciferase reporter and a Renilla luciferase normalization control. Twenty-four hours post transfection, 293T were stimulated for 16 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, co-stimulated with IFNβ and BMP9, or left unstimulated, followed by cell lysis and a Dual-luciferase assay readout. ( B ) Results from the Dual-luciferase assay with the BRE-Luciferase (top panel) and MX1-Luciferase (bottom panel) reporter. ( C ) Cell lysates from ( B ) were analyzed by immunoblot for the expression of M27, US18 and US20 with a V5-specific antibody, β-Actin served as loading control. ( D ) 293T were co-transfected with expression plasmids for the BRE-Luciferase and Renilla reporters as in ( A ), together with V5-tagged US18, US20 or co-transfected with US18 and US20 in combination. 24 h post transfection 293 T were stimulated for 16 h with BMP9 (3 nM), or left unstimulated, followed by cell lysis and a Dual-luciferase assay readout. ( E ) 293T were co-transfected with expression plasmids for either Cherry-STING and cGAS-GFP (left panel), RIG-I N (middle panel), or IRF3-5D (a constitutively activate IRF3 mutant; right panel), together with the murine IFNβ-luciferase reporter (IFNβ-Luc) and the Renilla reporter as normalization control. Cells were additionally transfected with expression plasmids for EV, V5-tagged M35 (a known inhibitor of PRR-mediated signaling pathways, (Chan et al, ), US18 or US20. Twenty hours post-transfection, cells were lysed and a dual-luciferase assay was performed. Immunoblot analysis of cell lysates from the respective experiments for M35, US18, and US20 detected with a V5-specific antibody, and β-Actin as a loading control, are shown below. Data information: ( B – E ) Data are combined from three independent experiments, for the immunoblots one representative is shown. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Control, Lysis, Western Blot, Mutagenesis, Protein-Protein interactions, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) HFF-1 with doxycycline-inducible expression of US18-V5, US20-HA, or US18-V5 and US20-HA with corresponding control cell lines (EV1, EV2, EV1 + 2) were generated. Protein expression was induced with 1 µg/ml doxycycline for 20 h prior stimulation and verified by immunoblot with V5- and HA-specific antibodies, and β-Actin as a loading control. ( B ) HFF-1 US18-V5, HFF-1 US20-HA, or HFF-1 US18-V5 US20-HA were left untreated or protein expression was induced with 1 µg/ml doxycycline to the medium for 20 h. Cell lysates were either left untreated or treated with PNGase F for 3 h at 37 °C, followed by immunoblot analysis with V5-, HA-, and Calnexin-specific antibodies. ( C , D ) Indicated HFF-1 lines were treated with 1 µg/ml doxycycline to induce protein expression for 18 h, followed by 2 h of serum starvation. ( C ) Cells were stimulated for 1 h with BMP9 (3 nM), followed by cell lysis and immunoblot analysis with phospho-SMAD1/5/9, SMAD1, V5, HA, and Calnexin antibodies. ( D ) Cells were stimulated for 6 h with either IFNβ (5 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9, followed by RNA extraction from cell lysates and RT-qPCR for Id3 and Stat2 transcripts. Data information: ( A – C ) Experiment was performed three independent times, one representative is shown. ( D ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Expressing, Control, Generated, Western Blot, Lysis, RNA Extraction, Quantitative RT-PCR, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) Schematic representation of the workflow. Recombinant HCMV US18stop, HCMV US20stop, and HCMV US18/20stop were constructed by introducing a 16 base pair (bp) stop cassette within the respective coding region. HFF-1 were infected by centrifugal enhancement with HCMV WT, HCMV US18stop, HCMV US20stop, or HCMV US18/20stop (MOI 4 for the 3 h time point, MOI 0.5 for the 48 h time point). Three or 48 h post infection, cells were stimulated with BMP9 (3 nM) for (1) 2 h, followed by cell lysis and immunoblot analysis, or (2) 6 h, followed by RNA extraction from cell lysates and RT-qPCR. ( B , C ) Cell lysates from ( A ) were subjected to immunoblot analysis with p-SMAD1/5/9, SMAD1, HCMV UL35, and GAPDH-specific antibodies (top panel), and transcript levels of Id3 and Stat2 are shown below. Data information: ( B , C ) Data are combined from three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Recombinant, Construct, Infection, Lysis, Western Blot, RNA Extraction, Quantitative RT-PCR, Two Tailed Test

Journal: EMBO Reports

Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

doi: 10.1038/s44319-024-00072-2

Figure Lengend Snippet: ( A ) HFF-1 were infected by centrifugal enhancement with HCMV WT or HCMV US18/20stop (MOI 4). Three hours post infection, cells were stimulated with IFNβ (1 ng/ml) or BMP9 (3 nM) alone, or co-stimulated with IFNβ and BMP9 for 6 h, followed by RNA extraction from cell lysates and RT-qPCR. ( B – D ) Transcript levels of Id3 ( B ), the ISGs Irf9 and Stat2 ( C ), and HCMV transcripts HCMV IE1 and HCMV UL44 ( D ). Data information: Three independent experiments with similar results were performed and data shown is combined from two of the three independent experiments. Student’s t test (unpaired, two-tailed), n.s. not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± SD. .

Article Snippet: Recombinant human BMP4 (#314-BP-010), human BMP6 (#507-BP-020), human BMP9 (#3209-BP-010), human BMP15 (#5096-BM-005), human Activin B (#659-AB-005), and the human/mouse anti-BMP9 antibody (#AF3209) were purchased from R&D Systems.

Techniques: Infection, RNA Extraction, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: Expression, activity, and purification of recombinant BMP9. A , schematic diagram of BMP9 production and processing. B , schematic diagram of the generation of HBMP9. A His tag ( filled star ) is introduced at the beginning of the mature BMP9. C , conditioned medium from HEK-EBNA cells transfected with prepro-BMP9 ( lane 1 ) or prepro-HBMP9 ( lane 2 ) were separated on a non-reducing SDS-PAGE and probed with anti-BMP9 antibody (MAB3209). The diagram on the right shows the schematic drawing of the BMP9 molecules. D denotes dimer on non-reducing SDS-PAGE, and M denotes monomer on SDS-PAGE. D- and M-forms migrate slower in the HBMP9 due to the addition of the His 6 tag at the N terminus of the mature ligand as depicted in B. D , HEK cell-produced BMP9 and HBMP9 have comparable activity with BMP9 from R&D Systems. Conditioned media containing pBMP9 (because it is very likely to be present as a prodomain bound complex in the conditioned media) or pHBMP9 were quantified by ELISA using R&D BMP9 as a standard and subjected to signaling assay using C2C12 cells transfected with ALK1. Plasmid containing Renilla was co-transfected with reporter plasmid containing BMP response element-luciferase, and the luciferase activity induced by BMP9 signaling was read using the Promega Dual-Luciferase system. E , purified pBMP9 and HBMP9 were fractionated on a 12% non-reducing SDS-PAGE and stained by Coomassie Blue. F , identical samples of R&D Systems BMP9, pBMP9, and HBMP9 were run in parallel on three 12% non-reducing SDS-PAGE, then blotted separately against the following: anti-BMP9 antibody ( left ), anti-BMP9 prodomain antibody ( middle ), and anti-His tag antibody ( right ). A single asterisk indicates a minor band that could not be seen on the SDS-PAGE in E but reacted very strongly with anti-BMP9 antibody. This may be a species of partially processed BMP9. Double asterisks indicate nonspecific carrier protein from R&D Systems BMP9. Pro , prodomain.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Expressing, Activity Assay, Purification, Recombinant, Transfection, SDS Page, Produced, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Luciferase, Staining

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: M-form BMP9 is a non-covalently linked dimer. A , HBMP9 was loaded onto a Superdex 75 10/30 gel filtration column pre-equilibrated in 50 m m Tris·HCl, pH 7.4, containing 150 m m NaCl. Non-reducing SDS-PAGE of the peak fractions (B3 to B5) revealed the D- and M-forms of HBMP9 co-elute under the same peak. The doublet in the M-form on SDS-PAGE was probably due to a partial reduction of intramolecular disulfide bonds. B , a representative of the HBMP9 crystal ( left ) and two examples of washed single crystals ran on a non-reducing SDS-PAGE ( right ) demonstrated that each single crystal contains a mixture of D- and M-forms of HBMP9. C , crystal structure of HBMP9 ( left ), colored according to the B-factors (spectrum, blue to white to red , from 20 to 100) with Cys-73 in two conformations shown in sticks . Electron density (2 F o − F c map at 1σ) clearly shows two conformations of Cys-73 ( right ). D , HBMP9 was overlaid with the published BMP9 structures (Protein Data Bank codes 1ZKZ and 4FAO , all colored as described in C ). In the semi-transparent schematic, type I receptor ALK1 is shown in yellow , and activin receptor type 2B is shown in cyan as in BMP9·ALK1·activin receptor type 2B complex (Protein Data Bank code 4FAO ).

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Filtration, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: M-form BMP9 can bind to ALK1 and prodomain as the D-form. A , M- and D- forms of HBMP9 co-migrate on native PAGE, and both can form complexes with ALK1 ECD. B , M- and D-forms of BMP9 co-migrate, both as free form and as prodomain-bound form. In A and B , bands 1 to 6 from native PAGE were cut out, boiled in 2× SDS-loading buffer for 20 min before loading onto a non-reducing SDS-PAGE to confirm the identities. For SDS-PAGE in A , two parts of the same gel are shown.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Clear Native PAGE, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: Intermolecular disulfide bond is not required for BMP9 signaling activity. A , conditioned media (6 μl) from HEK-EBNA cells transfected with transfection reagent alone ( lane 1 ), empty vector ( lane 2 ), pro-BMP9 ( lane 3 ), pro-HBMP9 ( lane 4 ), or pro-HBMP9 C73S ( lane 5 ) were fractionated on a 12% SDS-PAGE and probed with anti-BMP9 prodomain antibody. The intensities of bands were quantified using ImageJ, and the ratio of HBMP9 C73S to HBMP9 obtained. HBMP9 C73S concentration in the conditioned medium was normalized to HBMP9 using the above ratio. B , left : hPAECs were serum-restricted in EGM-2, 0.1% FBS overnight, and stimulated with HBMP9 or HBMP9 C73S (both 0.25–3 ng/ml) for 1 h. Cells were harvested, and total cell protein was immunoblotted with anti-pSmad1/5/8 antibody. Band intensities of pSmad1/5/8 blots were analyzed using ImageJ, corrected by ratios obtained from the α-tubulin blot, and normalized to a 0.25 ng/ml wild type sample. Data of the mean ± S.E. from three repeats are shown on the right. C , after quiescence overnight in EGM-2, 0.1% FBS, hPAECs were treated with 1 ng/ml of HBMP9 or HBMP9 C73S. Samples were harvested at 1, 2, 6, and 24 h, and immunoblotting was carried out as described in B . Band intensity of pSmad1/5/8 blots were analyzed using ImageJ, corrected by ratios obtained from the α-tubulin blot and normalized to a wild type 1-h treatment sample. Data of the mean ± S.E. from three repeats are shown on the right. NS , not significant.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Activity Assay, Transfection, Plasmid Preparation, SDS Page, Concentration Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: BMP9 is regulated by redox potential. Purified HBMP9 ( A ), pBMP9 ( B ), or BMP6 ( C ) were incubated at room temperature overnight with PBS alone or redox buffer containing 0.1 m m GSSG and 0–20 m m GSH. Proteins were then run on a non-reducing SDS-PAGE and detected by Coomassie Blue. M * is the fully reduced form of BMP9.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Purification, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: M-form BMP9 is more susceptible to redox-dependent proteolysis. HBMP9 ( A ) or pBMP9 ( B ) was incubated in PBS or redox buffer containing 0.1/1 m m GSSG/GSH (0.1/1) or 0.1/4 m m GSSG/GSH (0.1/4) at room temperature overnight. The following day, an aliquot of each treatment was subjected to limited trypsin digestion at 37 °C, 3 h for HBMP9 or overnight for pBMP9. Reactions were stopped by addition of SDS-loading buffer and boiling at 100 °C for 10 min. Cleavage was monitored by fractionating samples on a 12% non-reducing SDS-PAGE and Coomassie Blue staining.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Incubation, SDS Page, Staining

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent Proteolysis *

doi: 10.1074/jbc.M114.579771

Figure Lengend Snippet: M-form BMP9 is preferentially cleaved in serum and model of BMP9 regulation by redox-dependent proteolysis. A , pBMP9 (0.3 μg) was reconstituted into 10 μl of serum (10 healthy human controls), and equal aliquots of samples were taken at 0 h ( lane 1 ) and after overnight incubation at 37 °C ( lane 2 ). Samples were then fractionated on a 12% SDS-PAGE under non-reducing conditions and detected by anti-BMP9 antibody (AF3209). The D- and M-forms of BMP9 at 0 h and overnight were quantified using ImageJ, and the % BMP9 remaining after overnight incubation was calculated and plotted using GraphPad Prism. Data are mean ± S.E. ( n = 10). B , model of the regulation of BMP9 concentration by redox-dependent proteolysis.

Article Snippet: Anti-BMP9 antibody (MAB3209 and AF3209), anti-BMP9 prodomain antibody (AF3879), and control BMP9 were purchased from R&D Systems, Inc. Anti-His tag antibody (37–2900) was purchased from Invitrogen.

Techniques: Incubation, SDS Page, Concentration Assay