anti bmp4 biotin antibody  (R&D Systems)

Journal: mAbs

Article Title: Targeting RGMb interactions: Discovery and preclinical characterization of potent anti-RGMb antibodies blocking multiple ligand bindings

doi: 10.1080/19420862.2024.2432403

Figure Lengend Snippet: Anticipated epitopes of RGMb mAbs 2C11 and 5C10 and PD-L2-binding interface in relation to BMP2-, BMP4-, Neo1-, and PD-L2-binding regions of RGMb.

Article Snippet: The following antibodies and secondary reagents were used: anti-M13-HRP (Sino Biological; 11973 mm05T-H), anti-huIgG-HRP antibody (Invitrogen; 17166078), anti-muIgG-HRP (BioLegend; 405306), anti-rabbit-Fc-HRP (GenScript; A01856) anti-rat-HRP (BioLegend; 405405), anti-c-Myc antibody 9E10 with HRP conjugate (Santa Cruz Biotech; sc-40 hRP), anti-His-tag antibody (Abcam; AB-18184), anti-BMP4-biotin antibody (R&D Systems; BAM7572), anti-BPM-2-biotin antibody (Peprotech; 500P195BT), anti-muBMP4 antibody (R&D Systems; MAB50201), anti-huIgG-FITC (Biolegend; 410720), anti-muIgG-APC (Affymetrix eBioscience; 17-4210-82), anti-huIgG-APC (BioLegend; 410711), anti-huPD-L2 mouse IgG (ProSci Inc, RF16025), anti-huRGMb rabbit IgG (Boster Bio, A06984–1), anti-huRGMb (R&D Systems; AF3630/MAB3630), streptavidin-HRP (Thermo Scientific; 10571164), streptavidin Poly-HRP (Thermo Scientific; 10571164).

Techniques: Binding Assay

Journal: Biomaterials Research

Article Title: Derivation of Mesenchymal Stem Cells through Sequential Presentation of Growth Factors via Gelatin Microparticles in Pluripotent Stem Cell Spheroids

doi: 10.34133/bmr.0184

Figure Lengend Snippet: Bone morphogenetic protein-4 (BMP4)- and fibroblast growth factor 2 (FGF2)-induced differentiation of induced pluripotent stem cells (iPSCs) to mesenchymal stem cells (MSCs) in 2-dimensional (2D) conditions. (A) Schematic diagram depicting iPSC-to-induced MSC (iMSC) differentiation via mesoderm induction. (B) Images of iPSCs, mesoderm-induced cells, and iMSCs. Scale bar = 200 μm. (C) Quantitative real-time gene analysis (qPCR; top panel) and western blot (WB) analysis (bottom panel) of the mesoderm markers Brachyury and SNAI1 (Snail1), along with the pluripotency marker SOX2, at 4 d postdifferentiation. (D) qPCR (top panel) and WB analysis (bottom panel) of the MSC markers CD73, CD90, CD105, and CD44 at 11 d postdifferentiation. (E) Flow cytometry (fluorescence-activated cell sorting [FACS]) analysis of the MSC markers CD73, CD90, and CD44 in cells treated with either BMP4 only or BMP4 + FGF2. The qPCR data were normalized to 18S. The WB data were normalized to β-actin. All data represent results from 3 independent experiments, each conducted in triplicate. The data are presented as mean ± SD (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). Individual data points and significance levels are indicated in graphs. DMEM, Dulbecco’s modified Eagle medium; SR, serum replacement; hiPSCs, human iPSCs; mRNA, messenger RNA; GF, growth factor.

Article Snippet: The slow- and fast-degrading GelMPs were incubated with primary antibodies against FGF2 (Santa Cruz Biotechnology, USA) and BMP4 (PeproTech, USA) overnight and labeled with Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, USA) and Texas Red (Invitrogen, Thermo Fisher Scientific, USA) at 1 μl/100 μl of PBS, respectively.

Techniques: Western Blot, Marker, Flow Cytometry, Fluorescence, FACS, Modification

Journal: Biomaterials Research

Article Title: Derivation of Mesenchymal Stem Cells through Sequential Presentation of Growth Factors via Gelatin Microparticles in Pluripotent Stem Cell Spheroids

doi: 10.34133/bmr.0184

Figure Lengend Snippet: Gelatin microparticle (GelMP) fabrication, characterization, GF conjugation, and the release profiles of BMP4 and FGF2. (A) Scanning electron microscopy (SEM) images of fast-degrading (4 mM glutaraldehyde) and slow-degrading (12 mM glutaraldehyde) GelMPs. Scale bar = 100 μm. Size distributions of fast- and slow-degrading GelMPs. (B) Physical properties of fast- and slow-degrading GelMPs. (C) Representative immunofluorescent images of BMP4-conjugated fast-degrading GelMPs and FGF2-conjugated slow-degrading GelMPs. Scale bar = 100 μm. (D) Degradation rate of 5 mg of microparticles in collagenase (5 μg/ml) solution. (E) Cumulative release (%) of 1 μg of BMP4 from fast-degrading GelMPs and 1 μg of FGF2 from slow-degrading GelMPs incubated for 20 d in collagenase (5 μg/ml) solution. The obtained data represent results from 3 independent experiments, each conducted in triplicate. PBS, phosphate-buffered saline.

Article Snippet: The slow- and fast-degrading GelMPs were incubated with primary antibodies against FGF2 (Santa Cruz Biotechnology, USA) and BMP4 (PeproTech, USA) overnight and labeled with Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, USA) and Texas Red (Invitrogen, Thermo Fisher Scientific, USA) at 1 μl/100 μl of PBS, respectively.

Techniques: Conjugation Assay, Electron Microscopy, Incubation, Saline

Journal: Scientific Reports

Article Title: VIRMA promotes NSCLC progression by modifying ADAR m 6 A and increasing the activity of the TGF-β signaling pathway

doi: 10.1038/s41598-025-97237-3

Figure Lengend Snippet: The expression of TGF-β core genes in VIRMA overexpressed cell lines. ( A ) Protein (Western blotting analysis) levels of BMP2 in A549 and PC9 cells. ( B ) Protein (Western blotting analysis) levels of BMP4 in A549 and PC9 cells. ( C ) Protein (Western blotting analysis) levels of P-SMAD5 in A549 and PC9 cells. ( D ) Protein (Western blotting analysis) levels of P-SMAD3 in A549 and PC9 cells. ( E ) Protein (Western blotting analysis) levels of SMAD5 in A549 and PC9 cells. ( F ) Protein (Western blotting analysis) levels of SMAD3 in A549 and PC9 cells. ( G ) Protein (Western blotting analysis) levels of TGF-βIin A549 and PC9 cells. ( H ) Protein (Western blotting analysis) levels of TGF-βII in A549 and PC9 cells. ( I ) Protein (Western blotting analysis) levels of TGF-β1/2 in A549 and PC9 cells. ( J ) Western blot assay was employed to check the expression level of VIRMA in the overexpression experiments. GAPDH was used as a loading control. Statistical significance is denoted by * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Protein band intensity was assessed using ImageJ 1.46 (National Institutes of Health).The antibodies utilized in this research comprised VIRMA (ab271136, 1:1000, Abcam), ADAR (AF6153, 1:1000, Beyotime), TGF-β Receptor I (AF8148, 1:1000, Beyotime), TGF-β Receptor II (AF8151,1:1000, Beyotime), Smad5 (AF1405, 1:1000, Beyotime), Smad3 (AF1504, 1:1000, Beyotime), Phospho-Smad5 (Ser463/465) (AF1375, 1:1000, Beyotime), Phospho-Smad3 (Ser423/425) (AF1759, 1:1000, Beyotime), BMP2 (AF0075, 1:1000, Beyotime), and BMP4 (AF6312, 1:1000, Beyotime).

Techniques: Expressing, Western Blot, Over Expression, Control

Journal: Scientific Reports

Article Title: VIRMA promotes NSCLC progression by modifying ADAR m 6 A and increasing the activity of the TGF-β signaling pathway

doi: 10.1038/s41598-025-97237-3

Figure Lengend Snippet: The expression of TGF-β core genes in VIRMA silenced cell lines. ( A ) Protein (Western blotting analysis) levels of BMP2 in A549 and PC9 cells. ( B ) Protein (Western blotting analysis) levels of BMP4 in A549 and PC9 cells. ( C ) Protein (Western blotting analysis) levels of P-SMAD5 in A549 and PC9 cells. ( D ) Protein (Western blotting analysis) levels of P-SMAD3 in A549 and PC9 cells. ( E ) Protein (Western blotting analysis) levels of SMAD5 in A549 and PC9 cells. ( F ) Protein (Western blotting analysis) levels of SMAD3 in A549 and PC9 cells. ( G ) Protein (Western blotting analysis) levels of TGF-βIin A549 and PC9 cells. ( H ) Protein (Western blotting analysis) levels of TGF-βII in A549 and PC9 cells. ( I ) Protein (Western blotting analysis) levels of TGF-β1/2 in A549 and PC9 cells. ( J ) Western blot assay was employed to check the expression level of VIRMA in the knockdown experiments. GAPDH was used as a loading control. Statistical significance is denoted by * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Protein band intensity was assessed using ImageJ 1.46 (National Institutes of Health).The antibodies utilized in this research comprised VIRMA (ab271136, 1:1000, Abcam), ADAR (AF6153, 1:1000, Beyotime), TGF-β Receptor I (AF8148, 1:1000, Beyotime), TGF-β Receptor II (AF8151,1:1000, Beyotime), Smad5 (AF1405, 1:1000, Beyotime), Smad3 (AF1504, 1:1000, Beyotime), Phospho-Smad5 (Ser463/465) (AF1375, 1:1000, Beyotime), Phospho-Smad3 (Ser423/425) (AF1759, 1:1000, Beyotime), BMP2 (AF0075, 1:1000, Beyotime), and BMP4 (AF6312, 1:1000, Beyotime).

Techniques: Expressing, Western Blot, Knockdown, Control

Journal: Scientific Reports

Article Title: VIRMA promotes NSCLC progression by modifying ADAR m 6 A and increasing the activity of the TGF-β signaling pathway

doi: 10.1038/s41598-025-97237-3

Figure Lengend Snippet: The expression of TGF- β core genes in VIRMA-silenced cell lines along with ADAR plasmid overexpression. ( A ) Protein (Western blotting analysis) levels of BMP2 in A549 and PC9 cells. ( B ) Protein (Western blotting analysis) levels of BMP4 in A549 and PC9 cells. ( C ) Protein (Western blotting analysis) levels of P-SMAD5 in A549 and PC9 cells. ( D ) Protein (Western blotting analysis) levels of P-SMAD3 in A549 and PC9 cells. ( E ) Protein (Western blotting analysis) levels of SMAD5 in A549 and PC9 cells. ( F ) Protein (Western blotting analysis) levels of SMAD3 in A549 and PC9 cells. ( G ) Protein (Western blotting analysis) levels of TGF-βIin A549 and PC9 cells. ( H ) Protein (Western blotting analysis) levels of TGF-βII in A549 and PC9 cells. ( I ) Protein (Western blotting analysis) levels of TGF-β1/2 in A549 and PC9 cells. GAPDH was used as a loading control. Statistical significance is denoted by * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Protein band intensity was assessed using ImageJ 1.46 (National Institutes of Health).The antibodies utilized in this research comprised VIRMA (ab271136, 1:1000, Abcam), ADAR (AF6153, 1:1000, Beyotime), TGF-β Receptor I (AF8148, 1:1000, Beyotime), TGF-β Receptor II (AF8151,1:1000, Beyotime), Smad5 (AF1405, 1:1000, Beyotime), Smad3 (AF1504, 1:1000, Beyotime), Phospho-Smad5 (Ser463/465) (AF1375, 1:1000, Beyotime), Phospho-Smad3 (Ser423/425) (AF1759, 1:1000, Beyotime), BMP2 (AF0075, 1:1000, Beyotime), and BMP4 (AF6312, 1:1000, Beyotime).

Techniques: Expressing, Plasmid Preparation, Over Expression, Western Blot, Control

Journal: eLife

Article Title: Mutations that prevent phosphorylation of the BMP4 prodomain impair proteolytic maturation of homodimers leading to lethality in mice

doi: 10.7554/elife.105018

Figure Lengend Snippet: Figure 1. Point mutations predicted to interfere with phosphorylation of the BMP4 prodomain selectively interfere with BMP4 homodimer but not BMP4/7 heterodimer activity. Schematic illustrating sequential cleavage of BMP4 homodimers (A) and BMP4/7 heterodimers (B). BMP4 is sequentially cleaved at two sites to generate the mature ligand (light green) together with large (dark green) and small (yellow) prodomain fragments. (C) Sequence alignment of a portion of the prodomain of human (h), mouse (m), zebrafish (z), Xenopus (x), and chick (c) BMP4 to illustrate the conserved S-X-G FAM20C recognition motif. (D) RNA encoding wild type or point mutant forms of BMP4 were injected alone or together with BMP7 near the dorsal marginal zone (DMZ) of 4-cell embryos. DMZ and ventral marginal zone (VMZ) explants were isolated at stage 10 and pSmad1 levels were analyzed by immunoblot as illustrated. Blots were reprobed with actin as a loading control. Black bar indicates where a non-relevant intervening lane was removed using photoshop. (E, F) Quantitation of relative pSmad1 levels normalized to actin in at least three independent experiments (mean ± SD, data analyzed using an unpaired t-test). (G) RNA encoding wild type or point mutant forms of BMP4 were injected alone or together with BMP7 near the animal pole of 2-cell embryos. Ectodermal explants were isolated at stage 10 and tbxt levels were analyzed by semi-quantitative RT-PCR as illustrated. (H, I) Quantitation of relative tbxt levels normalized to odc in at least three independent experiments (mean ± SD, data analyzed using an unpaired t-test).

Article Snippet: Proteins were separated by electrophoresis on 10% or 12% gels and transferred to PVDF membranes that were probed with anti- Bmp4 (1:1000, Santa Cruz RRID:AB_2063534), anti- pSmad1/5 (1:1000, Cell Signaling, RRID:AB_491015), or anti- actin (1:10,000, Sigma, RRID:AB_476693) antibodies followed by HRP- conjugated anti- rabbit IgG or anti- mouse IgG2b (Jackson ImmunoResearch) secondary antibodies.

Techniques: Phospho-proteomics, Activity Assay, Sequencing, Mutagenesis, Injection, Isolation, Western Blot, Control, Quantitation Assay, Quantitative RT-PCR

Journal: eLife

Article Title: Mutations that prevent phosphorylation of the BMP4 prodomain impair proteolytic maturation of homodimers leading to lethality in mice

doi: 10.7554/elife.105018

Figure Lengend Snippet: Figure 4. Bmp4−/E93G mutants die during embryogenesis with defects in ventral body wall closure, small or absent eyes and heart defects. Photographs of E13.5 (A–C) or E14.5 (D–G) wild type (A, D) or mutant (B, C, E–G) littermates. The position of the liver inside (A–F) or outside (C, G) of the abdomen is indicated. Hematoxylin and eosin-stained coronal sections of hearts dissected from E14.5 wild type (H) or Bmp4−/E93G littermates (I). Asterisk denotes ventricular septal defect and arrowheads indicate thin, non-compacted ventricular wall (I). Scale bar applies across each row.

Article Snippet: Proteins were separated by electrophoresis on 10% or 12% gels and transferred to PVDF membranes that were probed with anti- Bmp4 (1:1000, Santa Cruz RRID:AB_2063534), anti- pSmad1/5 (1:1000, Cell Signaling, RRID:AB_491015), or anti- actin (1:10,000, Sigma, RRID:AB_476693) antibodies followed by HRP- conjugated anti- rabbit IgG or anti- mouse IgG2b (Jackson ImmunoResearch) secondary antibodies.

Techniques: Mutagenesis, Staining

Journal: eLife

Article Title: Mutations that prevent phosphorylation of the BMP4 prodomain impair proteolytic maturation of homodimers leading to lethality in mice

doi: 10.7554/elife.105018

Figure Lengend Snippet: Figure 6. E93G and S91C mutations lead to accumulation of BMP4 precursor protein and reduced levels of cleaved ligand and pSmad1 in vivo. Levels of pSmad1 and BMP4 were analyzed in protein lysates isolated from E10.5 Bmp4S91C (A–D) or Bmp4E93G (E–H) homozygotes, heterozygotes, or wild type littermates. Arrows in panels A and E denote a more slowly migrating BMP4 precursor protein band detected in lysates from mutant, but not wild type embryos. (I–P) Levels of pSmad1, BMP4 precursor protein, and cleaved BMP4 ligand were analyzed in cell lysates and conditioned media of mouse

Article Snippet: Proteins were separated by electrophoresis on 10% or 12% gels and transferred to PVDF membranes that were probed with anti- Bmp4 (1:1000, Santa Cruz RRID:AB_2063534), anti- pSmad1/5 (1:1000, Cell Signaling, RRID:AB_491015), or anti- actin (1:10,000, Sigma, RRID:AB_476693) antibodies followed by HRP- conjugated anti- rabbit IgG or anti- mouse IgG2b (Jackson ImmunoResearch) secondary antibodies.

Techniques: In Vivo, Isolation, Mutagenesis

Journal: eLife

Article Title: Mutations that prevent phosphorylation of the BMP4 prodomain impair proteolytic maturation of homodimers leading to lethality in mice

doi: 10.7554/elife.105018

Figure Lengend Snippet: Figure 7. BMP4E93G and BMP4S91C precursor proteins are O- and N-glycosylated and exit the ER. (A, B) Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 Bmp4S91C heterozygotes, Bmp4E93G homozygotes, or wild type littermates. Protein lysates were left untreated or were treated with O-glycosidase and α-neuraminidase to remove O-linked glycosylation (A) or with EndoH or PNGase to remove N-linked glycosylation (B). Immunoblots were probed with antibodies directed against BMP4 to detect BMP4 precursor protein. Bands corresponding to Endo H-sensitive (asterisks) and Endo H-resistant PNGase-sensitive BMP4 (arrowheads) are indicated in B.

Article Snippet: Proteins were separated by electrophoresis on 10% or 12% gels and transferred to PVDF membranes that were probed with anti- Bmp4 (1:1000, Santa Cruz RRID:AB_2063534), anti- pSmad1/5 (1:1000, Cell Signaling, RRID:AB_491015), or anti- actin (1:10,000, Sigma, RRID:AB_476693) antibodies followed by HRP- conjugated anti- rabbit IgG or anti- mouse IgG2b (Jackson ImmunoResearch) secondary antibodies.

Techniques: Isolation, Glycoproteomics, Western Blot

Journal: eLife

Article Title: Mutations that prevent phosphorylation of the BMP4 prodomain impair proteolytic maturation of homodimers leading to lethality in mice

doi: 10.7554/elife.105018

Figure Lengend Snippet: Figure 8. Hypothetical models for how prodomain phosphorylation regulates proteolytic maturation of BMP4. (A) Model 1: Phosphorylation of BMP4 by Fam20C within the trans-Golgi network (TGN) directs subcellular trafficking of BMP4 out of the Golgi to the cell surface via a route that is distinct from the pathway taken by furin. Furin- and BMP4-containing vesicles fuse in a region adjacent to the cell surface where furin cleaves BMP4

Article Snippet: Proteins were separated by electrophoresis on 10% or 12% gels and transferred to PVDF membranes that were probed with anti- Bmp4 (1:1000, Santa Cruz RRID:AB_2063534), anti- pSmad1/5 (1:1000, Cell Signaling, RRID:AB_491015), or anti- actin (1:10,000, Sigma, RRID:AB_476693) antibodies followed by HRP- conjugated anti- rabbit IgG or anti- mouse IgG2b (Jackson ImmunoResearch) secondary antibodies.

Techniques: Phospho-proteomics

Journal: Scientific Reports

Article Title: Extracellular vesicles derived from Msh homeobox 1 ( Msx1 )-overexpressing mesenchymal stem cells improve digit tip regeneration in an amputee mice model

doi: 10.1038/s41598-024-72647-x

Figure Lengend Snippet: Schematic representation of study design. ( A ) BlCs culture and Msx1 gene expression and characterization. ( B,C ) BlCs-EV isolation and characterization. ( D ) Creation of a non-regenerative digit tip. ( E ) BlCs-EV injection 4 DPA into the models. ( F ) The analysis of digit tip regeneration during 6 WPI. 4 DPA: 4 days post-amputation, 6 WPI: 6 weeks post-implantation.

Article Snippet: Then incubated overnight with primary antibodies against BMP4, FGF8, MSX1, and MSX2 proteins (Invitrogen) at 4 °C.

Techniques: Expressing, Isolation, Injection

Journal: Scientific Reports

Article Title: Extracellular vesicles derived from Msh homeobox 1 ( Msx1 )-overexpressing mesenchymal stem cells improve digit tip regeneration in an amputee mice model

doi: 10.1038/s41598-024-72647-x

Figure Lengend Snippet: BlCs culture, expansion, and Msx1 , Msx2 , Fgf8 , and Bmp4-related gene expression. ( A ) Fluorescent microscopic images of Msx1 (GFP + ) show Msx1 expression in BlCs immediately after culture and expansion (a), the nucleus of cells stained with DAPI dye (blue) (b), and illustrated the merged of a and b images ( n = 3) (c). ( B ) Real-time PCR analysis shows the Msx1 (a), Msx2 (b), Bmp4 (c), and Fgf8 (d) gene expression of BlCs in comparison with mBMSCs as a control group ( n = 3). Scale bar: 100 μm. Data are presented as means ± SD, (**** p < 0.001).

Article Snippet: Then incubated overnight with primary antibodies against BMP4, FGF8, MSX1, and MSX2 proteins (Invitrogen) at 4 °C.

Techniques: Expressing, Staining, Real-time Polymerase Chain Reaction, Comparison, Control

Journal: Scientific Reports

Article Title: Extracellular vesicles derived from Msh homeobox 1 ( Msx1 )-overexpressing mesenchymal stem cells improve digit tip regeneration in an amputee mice model

doi: 10.1038/s41598-024-72647-x

Figure Lengend Snippet: EVs isolation and characterization. ( A ) WB assessment indicated that the BlCs-EV were positive for CD81, a main marker of EVs. ( B ) Size and morphology of EVs obtained by SEM images, ( C ) Size of EVs observed using DLS (approximately 100–150 nm. ( D ) WB analysis of the EVs enriched proteins (BMP4, FGF8, and MSX1, MSX1). As full as possible length blots are presented in additional file 1: Figure Supplementary . The quantitative expression of the proteins MSX1, MSX2, FGF8, and BMP4 in BlCs-EVs and mBMSCs-EVs group ( n = 1). EVs: extracellular vesicle; WB: Western Blot; DLS: SEM: Scanning electron microscopy.

Article Snippet: Then incubated overnight with primary antibodies against BMP4, FGF8, MSX1, and MSX2 proteins (Invitrogen) at 4 °C.

Techniques: Isolation, Marker, Expressing, Western Blot, Electron Microscopy