polyclonal anti bmp2 bmp4 primary antibody  (R&D Systems)

Journal: Advanced therapeutics

Article Title: Bio-orthogonal, Site-Selective Conjugation of Recombinant Proteins to Microporous Annealed Particle Hydrogels for Tissue Engineering

doi: 10.1002/adtp.201900148

Figure Lengend Snippet: A) Representative live/dead cell viability image of human mesenchymal stem cells (hMSCs) within MAP hydrogels at 5 days. Average cell viability within MAP hydrogels was 83% ± 6% (n = 3). Scale bar = 200 μm. B) Representative hMSC spreading images within MAP hydrogels at 5 days overlayed with bright field of microparticles. Nuclear DAPI stain is represented in blue and cytoskeletal F-actin stain is represented in red. Average hMSC cell volume within MAP hydrogels was 11,200 μm3 ± 2,685 μm3 (n = 3). Scale bar = 200 μm. C) Schematic showing Tz-BMP2 functionalized MAP hydrogels with encapsulated hMSCs for a mineralization study. D) Tz-BMP2 and NF-BMP2 (5 μg/mL) functionalized MAP hydrogels compared using a modified surface ELISA (n = 3). Absorbance values demonstrate differences in ELISA development between NF- and Tz-BMP2 MAP hydrogels indicating differenes in BMP2 conjugation. The symbol ** indicates statistical significance between groups (p < 0.01). E) Comparison of MAP hydrogel-bound calcium mineralization after 21 days (n = 6 for BMP2 samples, n = 3 for media controls). Absorbance values show that only Tz-BMP2 functionalized MAP hydrogels induce hMSC osteogenic mineralization. The symbol * indicates statistical significance between all other experimental groups (p < 0.05).

Article Snippet: Mouse monoclonal, anti-human BMP2 antibody (Abcam, 100 μL, 0.1 μg mL −1 ) was incubated within each well for 3 hrs at room temperature.

Techniques: Staining, Modification, Enzyme-linked Immunosorbent Assay, Conjugation Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Hematopoietic TALE-Code Shows Normal Activity of IRX1 in Myeloid Progenitors and Reveals Ectopic Expression of IRX3 and IRX5 in Acute Myeloid Leukemia

doi: 10.3390/ijms23063192

Figure Lengend Snippet: Transcriptional regulation of IRX3 and IRX5. ( A ) RQ-PCR analysis of HOXA10 in AML cell lines ( left ). Transcript levels are indicated in relation to OCI-AML3, and IRX3/IRX5-positive cell lines are indicated in red. RQ-PCR analysis of OCI-AML3 ( above ) and MEGAL ( below ) showed downregulation of IRX3 and IRX5 after siRNA-mediated knockdown of HOXA10 ( right ). ( B ) RQ-PCR analysis of BMP2 in AML cell lines ( left ). Transcript levels are indicated in relation to OCI-AML3. ELISA results for BMP2 protein levels in AML cell line supernatants are inserted. RQ-PCR analysis of OCI-AML3 treated with BMP2 resulted in the upregulation of IRX3 and IRX5 ( right ). ( C ) RQ-PCR analysis of OCI-AML3 after siRNA-mediated knockdown of JUNB ( left ) and SMAD4 ( right ) showed downregulation of IRX3 and IRX5. ( D ) RQ-PCR analysis of JUNB in AML cell lines ( left ). Transcript levels are indicated in relation to OCI-AML3. RQ-PCR analysis of OCI-AML3 treated with inhibitory BMP2-antibody resulted in downregulation of JUNB, IRX3, and IRX5, while HOXA10 was not significantly affected ( right ). p -values are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significant).

Article Snippet: Cell treatments were performed for 20 h using 20 ng/mL of recombinant BMP2 (R & D Systems, Wiesbaden, Germany, #355-BM-010/CF) or 20 μg/mL of inhibitory monoclonal anti-BMP2 antibody (R & D Systems, #MAB3552).

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: The Hematopoietic TALE-Code Shows Normal Activity of IRX1 in Myeloid Progenitors and Reveals Ectopic Expression of IRX3 and IRX5 in Acute Myeloid Leukemia

doi: 10.3390/ijms23063192

Figure Lengend Snippet: Gene regulatory network of IRX1 and IRX3/IRX5 in AML. The figure summarizes the results of this study. TALE homeobox genes IRX1, IRX3, and IRX5 are located centrally, chromosomal aberrations lie upstream, and developmental TFs and BMP2-signaling components both upstream and downstream. Thus, these IRX genes are part of a regulatory gene network, controlling myeloid differentiation.

Article Snippet: Cell treatments were performed for 20 h using 20 ng/mL of recombinant BMP2 (R & D Systems, Wiesbaden, Germany, #355-BM-010/CF) or 20 μg/mL of inhibitory monoclonal anti-BMP2 antibody (R & D Systems, #MAB3552).

Techniques:

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a ) Fetal pericytes (CD146 + /CD31 − ) were infected with African strain ZIKV-MR766, Asian strains ZIKV-H/PF/2013 and PRVABC59 at MOI of 0.5 or treated with medium without virus (mock). Blue cell population is unstained control, red cell population is stained with CD146 and CD31 antibody. See for gating strategy. ( b ) At 1–4 and 8 dpi, cells were harvested for viral RNA analysis ( n =4). ( c ) Infectious virus titer was quantified in supernatants ( n =6 per group) collected from 1–4, 8 and 14 dpi. ( d ) At 3, 4 and 8 dpi, mature BMP2 protein from supernatants of mock- and ZIKV-infected pericytes was measured ( n =8). ( e ) Osteogenic gene expression in fetal pericytes was normalized to GAPDH and presented as fold changes relative to mock controls ( n =6 per group). ( f - g ) Alizarin red staining and Alizarin red concentration (mM) of ZIKV-infected fetal pericytes after 14 dpi ( n =4). Data are presented as mean ± SEM, using two-way ANOVA followed by Sidak’s multiple comparisons test ( e ) or one-way ( g ) ANOVA with Tukey’s posttest. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Data in d , e and g are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. Exact P values in g compared between Mock and ZIKV H/PF/2013 ( P= 0.0046) and between Mock and PRVABC59 ( P= 0.0030). Data are representative of three independent experiments.

Article Snippet: To inhibit BMP2/4 activity, cells were treated with 2 μg/mL of purified mouse IgG1 isotype control (Clone 11711, R&D Systems, #MAB002) or human BMP2/4 neutralizing antibody (Clone 100230, R&D Systems, #MAB3552100).

Techniques: Infection, Virus, Staining, Expressing, Concentration Assay

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a ) Schematic depicting the timeline for our ZIKV-vertical transmission mouse model. Time-mated immunocompetent hSTAT2 KI pregnant dams (8 to 12 weeks old) were i.p infected with PBS (mock control) or ZIKV PRVABC59 at mouse embryonic day 13.5 (E13.5) and pups were born between 19 to 21 days later. On post-delivery day (PD) 28, ZIKV-infected hSTAT2KI pups were sacrificed and brains were harvested for RNA extraction or cryo-sectioning. ( b ) Osteogenic gene expression in mock- ( n =3) or ZIKV-infected hSTAT2KI pup brains ( n =12) were normalized to GAPDH and presented as fold changes relative to mock controls. ( c ) At PD28, brain sections of pups were stained with DAPI (blue) and anti-NS2B antibody. ( d ) Brain sections were stained with Alizarin red for calcium deposit. Blue arrows indicate the locations of prominent calcium deposition. Data in b - d are representative of two independent experiments. Data in b are presented as mean ± SEM, using two-tailed unpaired Student t -test. * P < 0.05, ** P < 0.01 and **** P < 0.0001. Exact P values in b compared between PBS and ZIKV PRVABC59 ( Bmp2 P= < 0.0001; Runx2 P = 0.0027; Osx P = 0.0180; Alpl P= < 0.0001).

Article Snippet: To inhibit BMP2/4 activity, cells were treated with 2 μg/mL of purified mouse IgG1 isotype control (Clone 11711, R&D Systems, #MAB002) or human BMP2/4 neutralizing antibody (Clone 100230, R&D Systems, #MAB3552100).

Techniques: Transmission Assay, Infection, RNA Extraction, Expressing, Staining, Two Tailed Test

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: (A-B) U2OS cells were infected with ZIKV at an MOI of 0.5. ( a ) Supernatants from mock- and ZIKV-infected cultures were harvested for viral plaque assays ( n =8 biologically independent samples per group), and ( b ) cells were tested for viral RNA ( n =4 biologically independent samples per group). Data in b are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. ( c ) At 1–4 dpi, cell viability was determined by flow cytometry. See for gating strategy. ( d ) At 1–3 dpi, mock- and ZIKV-infected U2OS (MR766: MR; H/PF/2013: HPF) and their supernatants were collected, and TCA precipitated for immunoblot analysis with anti-BMP2 and Ponceau staining. ( e ) Phosphorylated SMAD1/5/9, total SMAD1 and ZIKV NS3 from whole cell lysates were detected by immunoblotting with indicated antibodies. ( f - h ) Inhibition of BMP2 activity. Mock- or ZIKV-infected U2OS cells were treated with 2 μg/mL of mouse IgG isotype control or human BMP2/4 neutralizing antibody. ( f ) At 4 dpi, cells were harvested for osteogenic gene expressions, normalized to GAPDH and presented as fold changes relative to mock controls ( n =3 biologically independent cells per group). ( g ) At 21 dpi, cells were stained with Alizarin red stain (calcium) and (h) Alizarin red stain concentration was quantified ( n =6). All results are representative of biological independent replicates from two independent experiments. Data are presented as mean ± SEM, using two-tailed unpaired Student t -test ( f ), two-way ANOVA followed by Sidak’s multiple comparisons test ( b ), or one-way ANOVA with Tukey’s posttest ( h ). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Exact P values in f compared between IgG and Nab group ( BMP2 P = 0.0034; RUNX2 P = 0.0267; OSX P = 0.0054; DMP1 P = 0.0113 and PDPN P = 0.0105). Significance differences of the means in h comparing between ZIKV-H/PF/2013 +IgG and ZIKV-H/PF/2013 +Nab group ( P= <0.0001)

Article Snippet: To inhibit BMP2/4 activity, cells were treated with 2 μg/mL of purified mouse IgG1 isotype control (Clone 11711, R&D Systems, #MAB002) or human BMP2/4 neutralizing antibody (Clone 100230, R&D Systems, #MAB3552100).

Techniques: Infection, Flow Cytometry, Western Blot, Staining, Inhibition, Activity Assay, Concentration Assay, Two Tailed Test

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a - b ) Mock- and ZIKV-infected U2OS cells were harvested at 2 and 4 dpi respectively for osteogenic gene expression. ( c ) Band intensity of pSMAD1/5/9 from U2OS whole cell lysate. ( d ) At day 1–4, IgG or nAb-treated and mock or ZIKV-infected U2OS cells ( n =6) were harvested for RNA extraction and viral load against ZIKV NS1 RNA was quantified using qRT-PCR (N.D.; Not detected) ( e ) Human primary fetal brain pericytes are infected with ZIKV PRVABC59 or PBS (mock control) that were treated with 2 μg/mL of mouse IgG isotype control or human BMP2/4 neutralizing antibody. At 4 dpi, cells were harvested for osteogenic gene expressions, normalized to GAPDH and presented as fold changes relative to mock controls ( n =4/group). Data in a , b, d and e are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. Data are analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparisons ( a ), Mann-Whitney U -test ( b ), or two-tailed unpaired Student t -test ( e ). * P < 0.05, ** P < 0.01, *** P < 0.001. Exact P values in b compared between Mock and ZIKV H/PF/2013 ( BMP2 P= 0.0002; RUNX2 P = 0.0011; OSX P = 0.0002; ALPL P = 0.0002; DMP1 P = 0.0002 and PDPN P = 0.0002). Exact P values in e compared between IgG-treated and Nab-treated group ( BMP2 P= 0.0054; RUNX2 P = 0.0005; OSX P = 0.0085; DMP1 P = 0.0099 and PDPN P = 0.0005). Data are representative of two independent experiments.

Article Snippet: To inhibit BMP2/4 activity, cells were treated with 2 μg/mL of purified mouse IgG1 isotype control (Clone 11711, R&D Systems, #MAB002) or human BMP2/4 neutralizing antibody (Clone 100230, R&D Systems, #MAB3552100).

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

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a ) Primary fetal pericytes transduced with lentivirus encoding individual ZIKV protein. Lenti-transduced cells were harvested for analysis of osteogenic gene expression normalized to GAPDH and expressed as fold changes relative to vector control ( n =8). ( b ) Schematic of ZIKV NS3 protease WT and H51A/S135A mutant (ZIKV NS3 Mut ). ( c ) Osteogenic gene expressions in WT and mutant (Mut) NS3-transduced pericytes ( n =4). Data in a and c are presented as mean ± SEM in box plots showing the upper (75%) and lower (25%) quartiles, with the horizontal line as the median and the whiskers as the maximum and minimum values observed. Data are presented as mean ± SEM, using two-tailed unpaired Student t -test. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values in c compared between WT and Mut NS3 group ( BMP2 P= 0.0012 ; RUNX2 P = 0.0013; OSX P = 0.0002; DMP1 P= 0.0030; PDPN P= <0.0001). Data are representative of three independent experiments.

Article Snippet: To inhibit BMP2/4 activity, cells were treated with 2 μg/mL of purified mouse IgG1 isotype control (Clone 11711, R&D Systems, #MAB002) or human BMP2/4 neutralizing antibody (Clone 100230, R&D Systems, #MAB3552100).

Techniques: Transduction, Expressing, Plasmid Preparation, Mutagenesis, Two Tailed Test

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a ) Sequence alignment comparison between BMP2 gene in human (homo sapiens; amino acid residues 1–396) and hamster (Cricetulus; amino acid residues 1–399). ( b ) Purity of BL21 strain-derived recombinant ZIKV NS3 protease, CHO-FD11 cells-derived full-length BMP2 and BMP4 were determined by coomassie blue stain. MW: molecular weight. ( c ) Sequence alignment comparison between human BMP2 (amino acid residues 1–396) and BMP4 (amino acid residues 1–408). ( d ) Sequence alignment of NS3 protease domain (amino acid residues 1–177) was compared across two African ZIKV (MR766, IbH30656) and two Asian ZIKV (PRVABC59 and H/PF/2103). The red-colored letters indicate protease catalytic triad. ( e ) In vitro cleavage assay of carboxyl terminal HA-tagged purified human BMP4 and purified ZIKV NS3 protease were performed at 37 °C for 3 h, followed by immunoblot analysis. Data in b and e are representative of three independent repeats.

Article Snippet: To inhibit BMP2/4 activity, cells were treated with 2 μg/mL of purified mouse IgG1 isotype control (Clone 11711, R&D Systems, #MAB002) or human BMP2/4 neutralizing antibody (Clone 100230, R&D Systems, #MAB3552100).

Techniques: Sequencing, Comparison, Derivative Assay, Recombinant, Staining, Molecular Weight, In Vitro, Cleavage Assay, Purification, Western Blot

Journal: Nature microbiology

Article Title: Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

doi: 10.1038/s41564-020-00850-3

Figure Lengend Snippet: ( a ) At 3 days after transfection with Flag-tagged WT or mutant NS3, the supernatants and lysates of U2OS cells were subjected to immunoblotting analysis or Ponceau staining. ( b ) U2OS stably-expressing WT or mutant NS3 were cultured and stained with Alizarin red. ( c ) Schematic of predicted BMP2 cleavage products. ( d ) In vitro cleavage assay of carboxyl terminal HA-tagged purified human BMP2 and purified ZIKV NS3 protease were performed at 37 °C for 3 h, followed by immunoblot analysis. ( e ) Cleaved BMP2 products from in vitro reaction were added to U2OS cells for 0 or 15 min and cells were harvested for immunoblot analysis. Data are representative of two independent experiments. ( f ) Schematic model of ZIKV NS3 protease-induced BMP2 maturation and osteogenic gene expression for fetal brain calcification. ZIKV NS3 protease efficiently cleaves pro-BMP2 to generate secreted mature BMP2 that induces its receptor-mediated osteogenic gene expression and calcification.

Article Snippet: To inhibit BMP2/4 activity, cells were treated with 2 μg/mL of purified mouse IgG1 isotype control (Clone 11711, R&D Systems, #MAB002) or human BMP2/4 neutralizing antibody (Clone 100230, R&D Systems, #MAB3552100).

Techniques: Transfection, Mutagenesis, Western Blot, Staining, Stable Transfection, Expressing, Cell Culture, In Vitro, Cleavage Assay, Purification

Journal: eLife

Article Title: Direct reprogramming of human smooth muscle and vascular endothelial cells reveals defects associated with aging and Hutchinson-Gilford progeria syndrome

doi: 10.7554/eLife.54383

Figure Lengend Snippet: From the left: panel one is BMP2, panel two is αSMA, panel three is DAPI and panel four is merge.

Article Snippet: Antibody , Anti-human BMP2 (Rabbit polyclonal) , Novus , Cat#nBP1-19751 RRID: AB_2227877 , IF(1:100).

Techniques:

Journal: eLife

Article Title: Direct reprogramming of human smooth muscle and vascular endothelial cells reveals defects associated with aging and Hutchinson-Gilford progeria syndrome

doi: 10.7554/eLife.54383

Figure Lengend Snippet: From the left: panel one is BMP2, panel two is αSMA, panel three is DAPI and panel four is merge.

Article Snippet: Antibody , Anti-human BMP2 (Rabbit polyclonal) , Novus , Cat#nBP1-19751 RRID: AB_2227877 , IF(1:100).

Techniques:

Journal: eLife

Article Title: Direct reprogramming of human smooth muscle and vascular endothelial cells reveals defects associated with aging and Hutchinson-Gilford progeria syndrome

doi: 10.7554/eLife.54383

Figure Lengend Snippet: Heat-map representing DE genes between reprogrammed vascular cells from young (N = 3) vs. old (N = 3) donors (iSMCs ( A ), iVECs ( B )). Z score = ± 1.5. DE genes with log 2 FC > 1 and FDR < 0.05. Quantification of BMP2 ( C ) and PALD1 ( D ) expression in human skin biopsies from young vs. old donors (N = 2 donors per condition; N = 10 tissue sections per condition; Student’s t-test with p<0.001 (***) and p<0.05 (*)). Representative images of BMP2 ( E ) and PALD1 ( F ) from skin biopsies obtained from young vs. old donors. Scale bars: 50 µm. Quantification of vascular permeability using young vs. old reprogrammed cells ( G–J ). Schematic showing the generation of an endothelial monolayer covering the interface with a 3D matrix embedding SMCs. Vascular permeability was quantified by measuring the variation of 70 kDa dextran fluorescent intensity across the interface ( G ). Representative images of dextran flow (red) through the endothelial monolayer in presence of young vs. old reprogrammed cells. Scale bar: 100 µm ( H ). Quantification of vascular permeability in presence of young vs. old iVECs (I, at least N = 40 independent measurements per condition in three biological replicates; ANOVA with Holm-Sidak test; comparison with primary VECs (** is p<0.01 and *** is p<0.001); comparison with young iVECs (♦♦♦is p<0.001); comparison with old iVECs (••• is p<0.001)) or young vs. old iSMCs (J, at least N = 40 independent measurements per condition in three biological replicates; ANOVA with Holm-Sidak test; * is p<0.05, ** is p<0.01 and *** is p<0.001). Quantification of vascular permeability in presence of PALD1 KD ( K ) or BMP2 KD ( L ) in primary VECs and SMCs, respectively (at least N = 60 independent measurements per condition in three biological replicates for each KD experiment; Student's t-test; *** is p<0.001).

Article Snippet: Antibody , Anti-human BMP2 (Rabbit polyclonal) , Novus , Cat#nBP1-19751 RRID: AB_2227877 , IF(1:100).

Techniques: Expressing, Permeability, Comparison

Journal: eLife

Article Title: Direct reprogramming of human smooth muscle and vascular endothelial cells reveals defects associated with aging and Hutchinson-Gilford progeria syndrome

doi: 10.7554/eLife.54383

Figure Lengend Snippet: Note that BMP2 KD was validated using qPCR due to technical problems with any of the tested anti-BMP2 antibodies (Western Blot). The use of these antibodies resulted in multiple aspecific bands.

Article Snippet: Antibody , Anti-human BMP2 (Rabbit polyclonal) , Novus , Cat#nBP1-19751 RRID: AB_2227877 , IF(1:100).

Techniques: Western Blot

Journal: eLife

Article Title: Direct reprogramming of human smooth muscle and vascular endothelial cells reveals defects associated with aging and Hutchinson-Gilford progeria syndrome

doi: 10.7554/eLife.54383

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human BMP2 (Rabbit polyclonal) , Novus , Cat#nBP1-19751 RRID: AB_2227877 , IF(1:100).

Techniques: Transfection, Construct, Retroviral, Expressing, Isolation, Clinical Proteomics, Blocking Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Software