Bmpr 2

Manufactured by Santa Cruz Biotechnology

Sourced in United States

BMPR-II is a cell surface receptor that belongs to the bone morphogenetic protein receptor family. It functions as a serine/threonine kinase and plays a role in signal transduction pathways involved in bone and cartilage development.

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7 protocols using bmpr 2

Immunofluorescence Staining of Stem Cells

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Immunofluorescence staining was performed as described [33 (link), 83 (link), 84 (link)]. Briefly, exponentially growing cells were fixed with 4% paraformaldehyde (PFA) for 10 min, washed with PBS and permeabilized with 1% NP-40 for 10min at room temperature. After being blocked with 10% donkey serum (Jackson Immuno-Research Laboratories, West Grove, PA) for 1h at room temperature, cells were incubated with various primary antibodies, including CD29, CD73, BMPRII, CD90, CD117/c-kit, CD105/endoglin, or BMPR-II antibody (all from Santa Cruz Biotechnology) for 1h at room temperature. Cells were washed with PBS and incubated with FITC-labeled secondary antibodies (Jackson ImmunoResearch Laboratories) for 30 min. DAPI (Invitrogen) was used to visualize nuclei. Stains were examined under a fluorescence microscope. Negative control cells were performed under the same conditions without primary antibodies. Representative images from at least three independent staining experiments are shown.

Hu X., Li L., Yu X., Zhang R., Yan S., Zeng Z., Shu Y., Zhao C., Wu X., Lei J., Li Y., Zhang W., Yang C., Wu K., Wu Y., An L., Huang S., Ji X., Gong C., Yuan C., Zhang L., Liu W., Huang B., Feng Y., Zhang B., Haydon R.C., Luu H.H., Reid R.R., Lee M.J., Wolf J.M., Yu Z, & He T.C. (2017). CRISPR/Cas9-mediated reversibly immortalized mouse bone marrow stromal stem cells (BMSCs) retain multipotent features of mesenchymal stem cells (MSCs). Oncotarget, 8(67), 111847-111865.

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Immunohistochemical Analysis of Ovarian Development

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Ovaries derived from gilts at age of 30–400 days were fixed in 10% (w/v) paraformaldehyde with 0.02 MPBS (pH 7.2) at 4°C for about 2 h. Next, they were cut into vertical slices of about 0.5 cm thickness and fixed in fresh 10% (w/v) paraformaldehyde for a cumulative period of 24 h. These slices were mounted in paraffin, and serially cut into 5 μm-thick sections by Rotary Microtome (MICROM, Germany), and stained with HE. Ovarian HE sections were observed and photographed under a fluorescent microscope (Zeiss, Germany).
Immunohistochemistry detection was performed by using the anti-Rabbit HRP-DAB Cell and tissue staining kit (R&D, CTS005) and anti-Goat HRP-DAB Cell and tissue staining kit (R&D, CTS008). Immunohistofluorescence examination was performed by using TSA plus Fluorescein (PerkinElmer, NEL741001KT) and Cyanine3.5 (PerkinElmer, NEL763001KT) kit. Antibodies of BMP15 (Eterlife, EL806306-100), GDF9 (Eterlife, EL901942-100), FSHR (Eterlife, EL912710), luteinizing hormone receptor (LHR) (Eterlife, EL904141), Caspase3 (Abcam, ab13847), 3βHSD (Abcam, ab154385), p-Smad1/5 (CST, 9516), Ki67 (Abcam, ab15580) and LC3B (Arigo, ARG55799) were diluted 1:100 with PBS. Other antibodies including ALK6 (Santa Cruz, sc5679), BMPR2 (Santa Cruz, sc5683), Smad2/3 (Santa Cruz, sc8332), Smad1/5/8 (Santa Cruz, sc6031R), and p-Smad2/3 (Santa Cruz, sc11769) were diluted 1:50 in PBS.

Qin Y., Tang T., Li W., Liu Z., Yang X., Shi X., Sun G., Liu X., Wang M., Liang X., Cong P., Mo D., Liu X., Chen Y, & He Z. (2019). Bone Morphogenetic Protein 15 Knockdown Inhibits Porcine Ovarian Follicular Development and Ovulation. Frontiers in Cell and Developmental Biology, 7, 286.

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Immunocytochemical Analysis of BMP4 and Germ Cell Markers

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The expression of BMP4 and its receptors as well as germ cell-specific markers in EB cells was obtained by immunocytochemistry according to the procedure described previously (Liu et al. 2015) (link). Briefly, iPS cells were fixed in 4% paraformaldehyde (PFA) and permeabilized with 0.4% Triton X-100. The cells were blocked with 10% serum and incubated with primary antibodies against BMP4 (Abcam), BMPR1A (Santa Cruz), BMPR1B (Santa Cruz), BMPR2 (Santa Cruz), PRDM1 (Abnova, Taipei, Taiwan) and VASA (Abcam) overnight at 4°C. The detailed information on antibodies was shown in Table 3. The cells were then incubated by goat anti-rabbit Alexa Fluor 594 (red)-labeled secondary antibody (Invitrogen) for 1 h. DAPI was used to label cell nuclei, and images were captured with a fluorescence microscope (Leica).

Yang S., Yuan Q., Niu M., Hou J., Zhu Z., Sun M., Li Z, & He Z. (2017). BMP4 promotes mouse iPS cell differentiation to male germ cells via Smad1/5, Gata4, Id1 and Id2. Reproduction (Cambridge, England), 153(2).

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Immunofluorescence Staining of Mesenchymal Stem Cells

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Immunofluorescence staining was performed 43, 51. Briefly, cells were seeded in 24‐well plates overnight, fixed with 4% paraformaldehyde, permeabilized with 1% NP‐40 and blocked with 10% donkey serum (Jackson Immuno‐Research Laboratories, West Grove, PA, USA), followed by incubating with CD73, CD105/endoglin, CD90/Thy‐1, CD166/ALCAM, BMPR‐II, CD117/c‐kit or CD29/Integrin β1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 hr at room temperature, as previously reported 43, 56. After being washed, cells were incubated with Texas Red or FITC labelled secondary antibody (Jackson ImmunoResearch Laboratories) for 30 min. Cell nuclei were counterstained with DAPI. Stains without primary antibodies were used as negative controls. Fluorescence images were recorded under an inverted fluorescence microscope.

Song D., Zhang F., Reid R.R., Ye J., Wei Q., Liao J., Zou Y., Fan J., Ma C., Hu X., Qu X., Chen L., Li L., Yu Y., Yu X., Zhang Z., Zhao C., Zeng Z., Zhang R., Yan S., Wu T., Wu X., Shu Y., Lei J., Li Y., Zhang W., Wang J., Lee M.J., Wolf J.M., Huang D, & He T. (2017). BMP9 induces osteogenesis and adipogenesis in the immortalized human cranial suture progenitors from the patent sutures of craniosynostosis patients. Journal of Cellular and Molecular Medicine, 21(11), 2782-2795.

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Comprehensive Protein Expression Analysis

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Western blot analyses for TLR4, total Caspase 3, and cleaved Caspase 3 (Cell Signaling Technology, Danvers, MA, USA; 1:1000), Ang2 (1:500; Sigma, St. Louis, MO, USA), TGFβ, NFkB, TNFα, IL-10, IL-1β, IL-4, Vegf, eNos, Vegf-D, BmpRII, and Vinculin (1:500; SantaCruz, Dallas, TX, USA) were performed, as previously described [24 (link)], by loading 30 µg of lung protein, followed by immunoblotting with the above antibodies and visualizing with Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Densitometric quantification was performed using ImageJ after normalizing with Vinculin, the loading control housekeeping protein.

Das P., Acharya S., Prahaladan V.M., Kumova O.K., Malaeb S., Behera S., Agarwal B., Christensen D.J., Carey A.J, & Bhandari V. (2021). Chitin-Derived AVR-48 Prevents Experimental Bronchopulmonary Dysplasia (BPD) and BPD-Associated Pulmonary Hypertension in Newborn Mice. International Journal of Molecular Sciences, 22(16), 8547.

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Immunoprecipitation and Western Blot Analysis

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For immunoprecipitation, the samples were collected and lysed in 50 mmol/L of Tris-HCl (pH 7.4) lysis buffer. The supernatant was immunoprecipitated with 5 μg of anti-BMPR-II (Cat. sc-393304; Santa Cruz, USA), anti-FLAG (Cat. F3165; Sigma), anti-Myc (Cat. 05-724; Millipore, USA) or anti-SMOC1 (Cat. ab155776; Abcam) overnight accordingly and then was incubated with Dynabeads™ Protein A (Cat. 1002 D, Invitrogen) for another 3 h at 4°C. Next, the cell lysates were subjected to western blot analysis as described previously.² (link) The primary antibodies used were SMOC1 (Cat. ab155776; Abcam), RUNX2 (Cat. 12556; Cell Signaling Technology), BMP2 (Cat. ab14933; Abcam), p-Smad1/5 (Cat. 9516; Cell Signaling Technology), Smad1 (Cat. 6944; Cell Signaling Technology), p-p38 (Cat. 4511; Cell Signaling Technology), p38 (Cat. 9212; Cell Signaling Technology), Caspase-3 (Cat. 9662; Cell Signaling Technology), BMPR-II (Cat. sc-393304; Santa Cruz), FLAG (Cat. F3165; Sigma) and Myc (Cat. 05-724; Millipore). Image Lab™ software was used to quantify the band density.

Wang Y., Gu J., Du A., Zhang S., Deng M., Zhao R., Lu Y., Ji Y., Shao Y., Sun W, & Kong X. (2021). SPARC-related modular calcium binding 1 regulates aortic valve calcification by disrupting BMPR-II/p-p38 signalling. Cardiovascular Research, 118(3), 913-928.

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Esophageal Cancer Cell Signaling Pathway

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Recombinant human BMP-2 was purchased from DaeWoong Pharmaceutical (Seoul, South Korea). Cell cycle related protein antibodies (cyclin D1, CDK4, p21, p53, p-Smad, and CDK6) and Hippo signaling pathway related protein antibodies (Mst1, Mst2, Sav1, LATS1, p-LATS1, Mob1, p-Mob1, YAP, and p-YAP) were obtained from Cell Signaling Technology (Danvers, MA, USA). The BMP-2 antibody was obtained from Abcam (Cambridge, UK) and BMPR II, RASSF1, Akt, p-Akt, TP63, and β-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). RASSF1 small interfering RNA (siRNA), YAP siRNA, or control siRNA were purchased from Santa Cruz biotechnology (Dallas, TX, USA). The human TE-8 and TE-12 esophageal cancer cell lines were obtained from Dr. Izzo (Unversity of Texas M.D. Anderson Cancer Center, Houston, TX, USA). DMEM-F12 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco), 100 mg/ml streptomycin, and 100 IU/ml penicillin (Gibco) was used for cell medium. Cells were maintained under standard conditions at 37 °C in a 5% CO₂ humidified atmosphere.

Kim S.M., Ye S., Rah S.Y., Park B.H., Wang H., Kim J.R., Kim S.H., Jang K.Y, & Lee K.B. (2016). RhBMP-2 Activates Hippo Signaling through RASSF1 in Esophageal Cancer Cells. Scientific Reports, 6, 26821.

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