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Synthetic peptide (Human). Synthetic phosphopeptide derived from the region of the human Insulin Receptor that contains tyrosine 972 (as numbered according to Ebina, et al. (tyrosine 960 according to Ullrich, et al.).
Biological actions of insulin are mediated by the Insulin Receptor (IR), a receptor tyrosine kinase that regulates multiple signaling pathways through activation of a series of phosphorylation cascades. The IR is a heterotetrameric protein consisting of two ligand-binding alpha subunits and two beta subunits that each contain a tyrosine kinase domain. Insulin binding to the extracellular domain leads to autophosphorylation of the receptor and activation of the intrinsic tyrosine kinase activity, which allows appropriate substrates to be phosphorylated. Tyrosine 972 is in the juxtamembrane Asn-Pro- Glu-Tyr (NPEY) motif. Phosphorylation of IR tyrosine 972 is required for the binding and/or phosphorylation of the adapter protein Shc, the PTB domain, IRS-1, PI3 kinase, and the Suppressor of Cytokine Signaling (SOCS).
Our Abpromise guarantee covers the use of ab5678 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||1/100 - 1/500.|
|IP||Use at an assay dependent concentration. PubMed: 17264162|
|WB||Use a concentration of 0.1 - 1 µg/ml. Detects a band of approximately 110 kDa.|
Immunofluorescence analysis of Insulin Receptor (phospho Y972) was done on 70% confluent log phase MCF7 cells with insulin treatment (100nM for 5 min). The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Anti-Insulin Receptor (phospho Y972) antibody (ab5678) at 2µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488® Goat Anti-Rabbit IgG Secondary Antibody at a dilution of 1/400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor 594® Phalloidin. Panel d is a merged image showing membrane localization. Panel e is untreated MCF7 cells. Panel f shows no primary antibody control. The images were captured at 20X magnification.
Upregulation and Antibody-Peptide Competition. Extracts of CHO-T cells transfected with an insulin receptor-containing vector and left unstimulated (1) or stimulated with 50 nM insulin for 5 minutes (2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with the Anti-Insulin Receptor (phospho Y972) antibody (ab5678) for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphorylated peptide corresponding to the phosphopeptide immunogen (3), a generic phosphotyrosine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab')2 anti-rabbit IgG HRP conjugate. The data show that only the phosphopeptide corresponding to Insulin Receptor (phospho Y972) completely blocks the antibody signal, demonstrating the specificity of the antibody. The data also show up-regulation of the signal upon stimulation with insulin in this cell system.