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Af887

Manufactured by R&D Systems
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Sourced in United Kingdom

AF887 is a recombinant human BMP-2 (Bone Morphogenetic Protein 2) protein. It is an extracellular signaling molecule that plays a role in bone and cartilage development.

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Lab products found in correlation

Mab391, by R&D Systems (1 mentions) Chemidoc system, by Bio-Rad (1 mentions) Cytoseal 60, by Thermo Fisher Scientific (1 mentions) 3 3 diaminobenzidine dab substrate kit, by Abcam (1 mentions) Eclipse e400, by Nikon (1 mentions) Hematoxylin, by Thermo Fisher Scientific (1 mentions) Active β catenin clone 8e7, by Merck Group (1 mentions) Phenylmethylsulphonyl fluoride, by Merck Group (1 mentions) Nherf1, by Abcam (1 mentions) Ach3k9 14, by Merck Group (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Ab3452, by Abcam (1 mentions) Sc 1616, by Santa Cruz Biotechnology (1 mentions)

Spelling variants of this product

AF887-sp (2 citations)

3 protocols using af887

1

PAPP-A-Mediated IGFBP-4 Cleavage and IGF Signaling

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The PAPP-A-mediated cleavage of endogenous IGFBP-4 in ascites was further assessed by incubating ascites samples (n = 10) with 0.1 nM recombinant PAPP-A [19 (link)] or buffer at 37°C for 6 h. Ascites samples containing the highest amounts of intact IGFBP-4 were chosen. Buffer containing rhIGF-I (0 or 5 ug/l) or rhIGF-I (5 ug/l) and PAPP-A (0.1 nM) were incubated and served as controls. The reaction mixtures were immediately used for measurements of bioactive IGF using the KIRA bioassay. In addition, IGFBP-4, C-terminal and N-terminal IGFBP-4 were determined in the reaction mixtures using immunoassays as previously described [22 (link), 23 (link)]. Finally, to assess the intracellular signalling pathways initiated by IGF-IR activation, cell lysates from the IGF bioactivity measurements were separated by 4–15% SDS-PAGE and transferred to PVDF membranes. Levels of phosphorylation of the intracellular proteins Akt, mTOR and S6 were determined by probing the blots with anti-p-Akt antibody (AF887, R&D Systems, Abingdon, UK), anti-p-TOR antibody (AF1665, R&D Systems) and anti-p-S6 antibody (AF3918, R&D Systems). Total IGF-IR levels in the cell lysates were determined using anti-hIGF-IR antibody (MAB391, R&D Systems) and used as loading controls. Additionally, stain-free total protein quantitation using the ChemiDoc™ system (Bio-Rad) served as total protein loading control.
Thomsen J., Hjortebjerg R., Espelund U., Ørtoft G., Vestergaard P., Magnusson N.E., Conover C.A., Tramm T., Hager H., Høgdall C., Høgdall E., Oxvig C, & Frystyk J. (2015). PAPP-A proteolytic activity enhances IGF bioactivity in ascites from women with ovarian carcinoma. Oncotarget, 6(31), 32266-32278.
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2

Immunohistochemical Evaluation of Phospho-AKT and Active β-Catenin

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Sections were deparaffinized, antigens retrieved, and blocked as described above. Primary antibodies used were phospho-AKT (Ser 473) (AF887; 1:200; R&D Systems, Minneapolis, MN) and active β-catenin (clone 8E7, #05-665; 1:300; Millipore, Billerica, MA). After washing, sections were incubated in 3% H2O2 in PBS for 15 minutes to block endogenous peroxidase activity. Sections were then incubated with secondary antibodies for 1 hour at room temperature. Secondary antibodies were Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit (ab6721; 1:1,000; Abcam, Cambridge, MA) and (HRP)-conjugated goat anti-mouse (#7076; 1:1,000; Cell Signaling Technology, Danvers, MA). Protein was visualized using, 3,3' Diaminobenzidine (DAB) substrate kit according to manufacturer’s instructions (Abcam). Sections were counterstained with Hematoxylin, dehydrated, and mounted with Cytoseal60 (Thermo Scientific). Cells were visualized using Nikon Eclipse E400 (Tokyo, Japan), and images were captured at 40× or 100× total magnification using QCapture software (Surrey, BC, Canada).
Bilir B., Osunkoya A.O., Wiles WG I.V., Sannigrahi S., Lefebvre V., Metzger D., Spyropoulos D.D., Martin W.D, & Moreno C.S. (2015). SOX4 is essential for prostate tumorigenesis initiated by PTEN ablation. Cancer research, 76(5), 1112-1121.
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3

Western Blot Analysis of Protein Modifications

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AMSCs were plated in 6-well plates as described above. Cells were treated with SAHA and/or solvent (0.0005% DMSO and 0.05% ethanol) as described in figure legends. After 24 hours, cells were lysed in radio-immunoprecipitation buffer (150 mM NaCl, 50 mM Tris pH 7.4, 1 % sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma) and phenylmethylsulphonyl fluoride (Sigma). Lysates were cleared by centrifugation. Protein concentrations were determined by the DC Protein Assay (Bio-Rad). Proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking in 5% non-fat dry milk for 45 minutes at room temperature, primary antibodies were added overnight at 4°C, followed by secondary antibodies for 1 hour at room temperature. Proteins were visualized using an ECL+ detection kit. Primary antibodies used were: Actin (1:10,000; sc-1616; Santa Cruz), Ac-H3 (K9/14) (1:10,000; 06–599; Millipore), H4 (1:10,000; 04–858; Millipore), Ac-H4 (K4/7/11/15) (1:20,000; 06–866; Millipore), pS473-AKT (1:4,000; AF887; R&D), pT24-FOXO1 (1:2,000; 2599; Cell Signaling), and NHERF1 (1:1,000; Ab3452, Abcam).
Dudakovic A., Camilleri E.T., Lewallen E.A., McGee-Lawrence M.E., Riester S.M., Kakar S., Montecino M., Stein G.S., Ryoo H.M., Dietz A.B., Westendorf J.J, & van Wijnen A.J. (2015). Histone deacetylase inhibition destabilizes the multi-potent state of uncommitted adipose-derived mesenchymal stromal cells. Journal of cellular physiology, 230(1), 52-62.
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