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Analyses of Smad1 (pS463/pS465)/Smad8 (pS465/pS467) expression. Western blot analyses of Smad1 (pS463/pS465)/Smad8 (pS465/pS467) expression by Ramos cells (Left Panel) and human peripheral blood mononuclear cells (PBMC; Middle Panel). Lysates were prepared from Ramos cells (20 µg total protein/lane) or PBMC (1 million cells/lane) that were cultured overnight in media containing 0.1% FBS and then either not treated (C) or treated (T) with Bone Morphogenetic Protein 6 (BMP-6; R&D Systems, Cat. No. 507-BP; 500 ng/ml, 15 min, 37°C). The lysates were electrophoresed, transferred to a membrane and blotted using Purified Rat anti-Smad1 (pS463/pS465)/Smad8 (pS465/pS467) antibody (at 0.5, 0.25 and 0.125 µg/ml for Lanes 1, 2, 3, respectively), HRP Goat Anti-Rat Ig (Cat. No. 554017) and a chemiluminescent detection system. Phosphorylated Smad1 and Smad8 were identified as ~60 kDa bands by Western blotting. Flow cytometric analysis of Smad1 (pS463/pS465)/Smad8 (pS465/pS467) expressed by PBMC (Right Panel). PBMC were cultured overnight in media containing 0.1% FBS and then either not treated (dashed line histogram) or treated with BMP-6 (500 ng/ml, 15 min, 37°C; solid line histogram). Cells were fixed in 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049; 10 min, 37˚C) and permeabilized in BD Phosflow™ Perm Buffer III (Cat. No. 558050; 30 min, on ice). Cells were then stained with BD Phosflow™ PE Rat anti-Smad1 (pS463/pS465)/Smad8 (pS465/pS467) (Cat. No. 562509) and Alexa Fluor® 647 Mouse Anti-Human CD20 (Cat. No. 558054) antibodies. Fluorescence histograms showing Smad1 and Smad8 phosphorylation were generated for CD20-positive gated events with the forward and side-light scatter characteristics of intact cells using a BD FACSCanto™ II Flow Cytometer System. Note: For all analyses shown, PBMC were isolated from freshly drawn EDTA blood.
BD Pharmingen™ Purified Rat anti-Smad1 (pS463/pS465)/Smad8 (pS465/pS467)
BD Pharmingen™ Purified Rat anti-Smad1 (pS463/pS465)/Smad8 (pS465/pS467)
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Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
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Recommended Assay Procedures
In human PBMC, overnight serum starvation was found to be necessary for detection of a BMP-6-induced increase in Smad1/8 phosphorylation. Serum starvation for 1 hour following PBMC isolation was not sufficient to reduce basal levels of Smad1 (pS463/pS465)/Smad8(pS465/pS467). Some donor variation was observed in the reduction of basal phosphorylation by overnight serum starvation.
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- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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The N6-1233 monoclonal antibody specifically binds to the Smad1 protein phosphorylated at the Ser463/465 sites and the Smad8 protein phosphorylated at the Ser465/467 sites. Smad1 and Smad8 are ~60 kDa proteins and are members of the Smad superfamily. The Smad family members are divided into three subfamilies: receptor regulated Smads or R-Smads, including Smads1, 2, 3, 5, and 8; common partner Smad, or Co-Smad, including Smad4; and inhibitory Smads, or I-Smad, including Smads 6 and 7. Activation of Transforming Growth Factor-beta (TGF-beta) superfamily serine/threonine kinase receptors, such as TGF-beta and Bone Morphogenic Protein (BMP) receptors, leads to the phosphorylation of R-Smads at several sites. It has been shown that Ser463 and Ser465 of Smad1 are phosphorylated by BMP receptors. In B cells and pre-B cells, BMP-6 has been shown to induce Smad1/5/8 phosphorylation and inhibit cell proliferation. Phosphorylated R-Smads form complexes with Co-Smad and translocate into the nucleus to regulate transcription affecting a wide range of critical processes including cell-fate determination, proliferation, morphogenesis, differentiation and apoptosis. The inhibitory Smads inhibit this pathway through two potential mechanisms: either by preventing R-Smads from binding to their corresponding receptors and/or by competing with Smad4, the Co-Smad, from binding to R-Smads. This antibody may crossreact with Smad5 pS463/pS465 based on sequence homology.
Development References (6)
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Hogan BL. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev. 1996; 10(13):1580-1594. (Biology). View Reference
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Hoodless PA, Haerry T, Abdollah S, et al. MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell. 1996; 85(4):489-500. (Biology). View Reference
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Kretzschmar M, Doody J, Massagué J. Opposing BMP and EGF signalling pathways converge on the TGF-beta family mediator Smad1. Nature. 1997; 389(6651):618-622. (Biology). View Reference
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Kretzschmar M, Liu F, Hata A, Doody J, Massague J. The TGF-beta family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. Genes Dev. 1997; 11:984-995. (Biology). View Reference
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Whitman M. Smads and early developmental signaling by the TGFbeta superfamily. Genes Dev. 1998; 12(16):2445-2462. (Biology). View Reference
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Yang J, Davies RJ, Southwood M, et al. Mutations in bone morphogenetic protein type II receptor cause dysregulation of Id gene expression in pulmonary artery smooth muscle cells: implications for familial pulmonary arterial hypertension. Circ Res. 2008; 102(10):1212-1221. (Biology). View Reference
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