lipolysis bmprii fc human mouse 811 br 100 r d systems spr  (R&D Systems)

Journal: The FASEB Journal

Article Title: Prodomain processing controls BMP‐10 bioactivity and targeting to fibrillin‐1 in latent conformation

doi: 10.1096/fj.202401694r

Figure Lengend Snippet: FIGURE 4 The processed BMP-10 CPLX binds robustly to BMP receptors whereas the unprocessed dimer shows no receptor interaction. Sensorgrams of SPR interaction studies of soluble BMP-10 GF, processed BMP-10 CPLX, and unprocessed BMP-10 dimer flowed over immobilized BMP receptors. Soluble analytes were injected onto immobilized BMPRII, ALK-1, and ENG at concentrations ranging from 0 to 80 nM. KDs were calculated from three independent experiments (N = 3).

Article Snippet: To assess the binding affinity of BMP- 10 processing variants to BMP receptors, the human IgG1- Fc- fusion ectodomains of BMPRII (#811- BR- 100/CF, R&D Systems), ALK- 1 (#370- AL- 100/CF, R&D Systems) as well as ENG (#1097- EN- 025/CF, R&D Systems) were immobilized at 500 or 800 RUs on a CM5 chip via amine coupling.

Techniques: Injection

Journal: Neural Regeneration Research

Article Title: BMPRII + neural precursor cells isolated and characterized from organotypic neurospheres: an in vitro model of human fetal spinal cord development

doi: 10.4103/1673-5374.373669

Figure Lengend Snippet: Human fetal spinal cord BMPRII immunostaining at gestational ages of 15–16 weeks. Cryostat section (12 μm) of the human fetal spinal cord estimated to be at gestational ages of 15–16 weeks showing intense BMPRII + immunoreactivity (green) along the ventral surface, as well as the dorsal (sensory) and ventral (motor) horns. Insets A–G shown at higher magnification, scale bar: 50 μm in D; nuclei counterstain DAPI (blue). Staining was observed along the dorsal surface and the sensory and motor neurons in the dorsal and ventral horns. The dotted red line demarcates regions of higher intensity BMPRII-immunoreactivity in dorsal horns. Magenta marks the outer margin of higher-intensity BMPRII-immunoreactivity in the ventral funiculus, while orange demarcates the extent of the structure. (A) BMPRII + immunoreactivity in the dorsal horn (B) BMPRII + immunoreactivity in the ventral horn (C) BMPRII + immunoreactivity at the ventral funiculus. (D–F) Vascular tube-like structures showed strong immunoreactivity to BMPRII staining, while (G) shows an example of staining of the meninges surrounding the spinal cord. The main anatomical structures are identified by labeling. 5× magnification, scale bar: 200 μm. BMP: Bone morphogenetic protein; BMPRII: type-II BMP receptor; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The slides were then incubated with primary antibodies for 16 hours at 4°C and secondary antibodies for 2 hours at room temperature: anti-human BMPRII (polyclonal goat antiserum raised against the human extracellular domain, used at 10 μg/mL; R&D Systems, Stillwater, MN, USA, Cat# AF467, RRID: AB_355622), anti-CD34 (vascular lineage marker; monoclonal antibody (1:50; mouse IgG1), Bio-Rad Laboratories (formerly Serotec), Hercules, CA, USA, clone QBEND/10, RRID: AB_1125255; Hughes et al., 2000) and anti-CD39 (1:400; mouse IgG2b, vascular lineage marker; Sigma-Aldrich (formerly Chemicon), Spring, TX, USA, clone 5F2, RRID: AB_10615429; Chan-Ling et al., 2004) made up with 1% bovine serum albumin (BSA) in PBS to detect the presence of BMPRII protein and vascular cells.

Techniques: Immunostaining, Staining, Labeling

Journal: Neural Regeneration Research

Article Title: BMPRII + neural precursor cells isolated and characterized from organotypic neurospheres: an in vitro model of human fetal spinal cord development

doi: 10.4103/1673-5374.373669

Figure Lengend Snippet: BMPRII + hNPCs (red) can also be identified in the human fetal brain cortex (A–C) and are identified by a lack of vascular marker expression (D–H). Scattered BMPRII + putative hNPCs (white arrows) are present in the parenchyma of a second-trimester human fetal brain cortex (B), nuclei counterstain DAPI (blue; A), shown merged in (C). By virtue of lower expression, these cells are clearly distinct from BMPRII + cells in vascular tubes (yellow arrow). 63× magnification, scale bar: 20 μm. Quadruple-labeled immunofluorescence of an intact second-trimester spinal cord, utilizing antibodies against BMPRII (G) combined with two established vascular lineage markers (CD34; green; D) and ecto-ADPase CD39 (magenta; E), showing a colocalized, triple-labeled vascular segment (yellow arrow in H). BMPRII (red) is highly expressed by vascular cells (yellow arrow), while scattered BMPRII + single cells (putative hNPCs) negative for vascular markers are evident in the tissue parenchyma (e.g., white arrow in (H); merge of all images). Nuclear counterstain DAPI (blue; F) labels nuclei. 63× magnification, scale bar: 20 μm. BMP: Bone morphogenetic protein; BMPRII: type-II BMP receptor; DAPI: 4′,6-diamidino-2-phenylindole; hNPCs: human neural precursor cells.

Article Snippet: The slides were then incubated with primary antibodies for 16 hours at 4°C and secondary antibodies for 2 hours at room temperature: anti-human BMPRII (polyclonal goat antiserum raised against the human extracellular domain, used at 10 μg/mL; R&D Systems, Stillwater, MN, USA, Cat# AF467, RRID: AB_355622), anti-CD34 (vascular lineage marker; monoclonal antibody (1:50; mouse IgG1), Bio-Rad Laboratories (formerly Serotec), Hercules, CA, USA, clone QBEND/10, RRID: AB_1125255; Hughes et al., 2000) and anti-CD39 (1:400; mouse IgG2b, vascular lineage marker; Sigma-Aldrich (formerly Chemicon), Spring, TX, USA, clone 5F2, RRID: AB_10615429; Chan-Ling et al., 2004) made up with 1% bovine serum albumin (BSA) in PBS to detect the presence of BMPRII protein and vascular cells.

Techniques: Marker, Expressing, Labeling, Immunofluorescence

Journal: Neural Regeneration Research

Article Title: BMPRII + neural precursor cells isolated and characterized from organotypic neurospheres: an in vitro model of human fetal spinal cord development

doi: 10.4103/1673-5374.373669

Figure Lengend Snippet: Effects of tissue culture condition on organotypic reaggregate neurosphere phenotype. (A) A comparison of sphere size showed that uniform stratification significantly favored populations of medium size spheres (200–500 μm diameter, e.g., J, ) compared with small (< 200 μm diameter, e.g. F, ) and large spheres (> 500 μm diameter). *** P < 0.001. (B) The effects of high-density reaggregation compared with low-density aggregation in combination with the presence or absence of LIF were assayed for their effects on neurosphere stratification. Both high-density and LIF-containing culture conditions were significantly different from low-density spheres or those grown in the absence of LIF. (C–F) Immunocharacterization of organotypic reaggregate neurospheres. Labeling with βIII-tubulin (C, green) shows an outer layer of immunoreactivity typical of organotypic neurospheres, while being negative for GFAP (D, red). The inside of organotypic neurospheres shows heterogenous βIII-tubulin and GFAP labeling. Co-staining with BMPRII (E) confirmed that βIII-tubulin + cells are double positive; merge shown in (F), and white boxed areas in C–F are zoomed below each sphere to show the fine detail of expressing cells. Reaggregate neurospheres were also characterized by positive immunoreactivity for LIF (G) and LIFR (H) but with no obvious indication of stratification; whereas endogenous BMP (I) and BMPRII (J) (all green) were both localized to the cell surface layer. All nuclei were counterstained with DAPI (magenta or blue, as indicated). Scale bar in F and J is 25 μm, and in F (inset below), 12.5 μm. BMP: Bone morphogenetic protein; BMPRII: type-II BMP receptor; DAPI: 4′,6-diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; LIF: leukemia inhibitory factor; LIFR: LIF receptor; ORN: organotypic reaggregate neurosphere.

Article Snippet: The slides were then incubated with primary antibodies for 16 hours at 4°C and secondary antibodies for 2 hours at room temperature: anti-human BMPRII (polyclonal goat antiserum raised against the human extracellular domain, used at 10 μg/mL; R&D Systems, Stillwater, MN, USA, Cat# AF467, RRID: AB_355622), anti-CD34 (vascular lineage marker; monoclonal antibody (1:50; mouse IgG1), Bio-Rad Laboratories (formerly Serotec), Hercules, CA, USA, clone QBEND/10, RRID: AB_1125255; Hughes et al., 2000) and anti-CD39 (1:400; mouse IgG2b, vascular lineage marker; Sigma-Aldrich (formerly Chemicon), Spring, TX, USA, clone 5F2, RRID: AB_10615429; Chan-Ling et al., 2004) made up with 1% bovine serum albumin (BSA) in PBS to detect the presence of BMPRII protein and vascular cells.

Techniques: Comparison, Labeling, Staining, Expressing

Journal: Neural Regeneration Research

Article Title: BMPRII + neural precursor cells isolated and characterized from organotypic neurospheres: an in vitro model of human fetal spinal cord development

doi: 10.4103/1673-5374.373669

Figure Lengend Snippet: Characterization of fluorescence-assisted cell sorting-sorted BMPRII + hNPCs. (A–E) Sorted cells were plated, fixed, and subjected to immunofluorescence staining with antibodies to O4 (blue), GFAP (red), MAP2ab (green), and the nuclei counterstained with DAPI (magenta). Image (A) shows sorted cells at low magnification. Images in C through E are shown with polarized bright field and merged at a higher magnification in B (from the area of the white box in A). (F) A representative single-label FACS histogram plot of fluorescence intensity versus cell number for collected BMPRII + cells (green trace) and control (2° antibody only; red trace). “Collected” shows the fluorescence intensity gate used to collect the BMPRII + cells. (G) Sorted cells were plated and the proportion of each phenotype was quantified. A significant increase was found for the number of MAP2ab + cells from approximately 24% in control to 83% in collected. (H) Cells were characterized using antibodies against each indicated marker where ‘+’ indicates > 90% of the cells with neuronal morphology were positive and ‘–’ indicates < 10% of these cells were positive. Data are expressed as the mean ± SEM, and scale bars are as indicated. BMP: Bone morphogenetic protein; BMPRII: type-II BMP receptor; DAPI: 4′,6-diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; hNPCs: human neural precursor cells; MAP: microtubule-associated protein; NeuN: neuron-specific nuclear protein; PSA-NCAM: polysialylated-neural cell adhesion molecule.

Article Snippet: The slides were then incubated with primary antibodies for 16 hours at 4°C and secondary antibodies for 2 hours at room temperature: anti-human BMPRII (polyclonal goat antiserum raised against the human extracellular domain, used at 10 μg/mL; R&D Systems, Stillwater, MN, USA, Cat# AF467, RRID: AB_355622), anti-CD34 (vascular lineage marker; monoclonal antibody (1:50; mouse IgG1), Bio-Rad Laboratories (formerly Serotec), Hercules, CA, USA, clone QBEND/10, RRID: AB_1125255; Hughes et al., 2000) and anti-CD39 (1:400; mouse IgG2b, vascular lineage marker; Sigma-Aldrich (formerly Chemicon), Spring, TX, USA, clone 5F2, RRID: AB_10615429; Chan-Ling et al., 2004) made up with 1% bovine serum albumin (BSA) in PBS to detect the presence of BMPRII protein and vascular cells.

Techniques: Fluorescence, FACS, Immunofluorescence, Staining, Control, Marker

Journal: Biomaterials advances

Article Title: Enhancing BMP-2-mediated osteogenesis with a synthetic heparan sulfate mimetic.

doi: 10.1016/j.bioadv.2023.213671

Figure Lengend Snippet: Fig. 2. Glycomimetic 15 binds BMP-2 and several other pro-regenerative factors with high affinity. A – SPR solution competition dosing experiments were performed with Ai - BMP-2, Aii - BMP-4, Aiii - BMP-7, Aiv BMPRII Fc chimera and Av - VEGF165 using a dose range of glycomimetic 15 (3.91 to 8000 nM). Avi – normalized response data extrapolated from sensorgrams Ai-Av. B – fluorescence polarization solution competition with BMP-2 (50 nM), 488-labelled heparin dp24 (0.5 nM) and increasing concentrations of glycomimetic 15 (red) or glycomimetic 18 (blue). Ci – DSF melt curves. Cii – first derivative of melt curve data. Ciii – BMP-2 melt temperature when incubated with multiple concentrations of glycomimetic 15, derived from the first derivative of melt curve data. Di – Normalized anti-factor Xa activity for glycomimetic 15 (black), heparin dp12 (red) and heparin dp24 (blue). Dii – anti-factor Xa activity in IU/mg, calculated using USP heparin standard curve. Bars represent mean, and error bars represent S.D. All data is representative of three independent experiments.

Article Snippet: BMP-2 (cat. # 355-BM/ CF), BMP-4 (cat. # 314-BP/CF), BMP-7 (cat. # 354-BP/CF), BMPRII Fc chimera (cat. # 811-BR), Noggin (cat. # 6057-NG/CF), VEGF165 (cat. # 293-VE/CF) polyclonal Goat anti-Human BMP-2/BMP-4 IgG (cat. # AF355), biotinylated monoclonal Mouse anti-Human BMP-2 IgG (cat. #BAM3552) were purchased from R&D systems.

Techniques: Fluorescence, Incubation, Derivative Assay, Activity Assay

Journal: Biomaterials advances

Article Title: Enhancing BMP-2-mediated osteogenesis with a synthetic heparan sulfate mimetic.

doi: 10.1016/j.bioadv.2023.213671

Figure Lengend Snippet: Fig. 5. Glycomimetic 15 influences BMPRII: BMP-2 and BMP-2: cell surface interactions. Ai – Co-immunoprecipitation and Western blot of BMP-2 (125 nM) with BMPRII Fc chimera (250 nM) ± increasing concentrations of either glycomimetic 15 or 18. Aii – Log2 fold change of densitometry values obtained from three in dependent co-immunoprecipitation experiments – bars represent mean log2 fold change ±95 % CI, normalized to BMPRII + BMP-2 alone. Bi – Flow cytometry histograms of C2C12 cells stained with BMP-2 (black histogram) ± increasing concentrations of glycomimetic 15 (red histogram) or glycomimetic 18 (blue histo gram). Bii – Cell surface binding of BMP-2 to C2C12 cells incubated with increasing concentrations of glycomimetic 15 (red) or glycomimetic 18 (blue) – data is mean geometric mean fluorescence ± S.D. normalized to BMP-2 alone. All data either constitutes (Aii, Bii) or is representative of (Ai, Bi) three independent experiments. Statistical analysis was performed via one-way ANOVA with Tukey’s post hoc testing (Aii) or two-way ANOVA with ˇSíd´ak or Bonferroni post hoc testing (Bii). Sig nificance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Article Snippet: BMP-2 (cat. # 355-BM/ CF), BMP-4 (cat. # 314-BP/CF), BMP-7 (cat. # 354-BP/CF), BMPRII Fc chimera (cat. # 811-BR), Noggin (cat. # 6057-NG/CF), VEGF165 (cat. # 293-VE/CF) polyclonal Goat anti-Human BMP-2/BMP-4 IgG (cat. # AF355), biotinylated monoclonal Mouse anti-Human BMP-2 IgG (cat. #BAM3552) were purchased from R&D systems.

Techniques: Immunoprecipitation, Western Blot, Flow Cytometry, Staining, Binding Assay, Incubation, Fluorescence