Stimulation and Antibody-Peptide Competition. Lysates of NIH3T3 cells untreated (1) or treated with 50ng/mL PDGF for 15 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 and then incubated with the Gab1 [pY627] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide 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 and quote;)2 anti-rabbit IgG HRP conjugate (Cat. no. ALI4404) and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to Gab1 [pY627] blocks the signal, demonstrating the specificity of the antibody. The data also show the induction of Gab1 [pY627] phosphorylation by the addition of PDGF in this cell system.
|Tested species reactivity||Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human Gab1 that contains tyrosine 627. The sequence is conserved in mouse.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 50% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Western Blot (WB)||Assay Dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Gab1 is a 115 kDa multiple docking protein that plays an essential role in cellular growth, transformation and apoptosis. Gab1 can be phosphorylated by multiple receptor tyrosine kinase (RTKs), including: insulin receptor (IR), platelet-derived growth factor receptor beta] (PDGFRbeta]), hepatocyte growth factor/scatter factor receptor (HGFR/SFR or c-Met), and epidermal growth factor receptor (EGF), as well as in response to cell-cell adhesion. Gab1 is tyrosine phosphorylated on at least 16 sites, some of which serve as binding sites for phosphoatidylinositol 3-kinase (PI3K), Grb2, PLC gamma 1, Nck, and SHP2. Phosphorylation of Gab1 on tyrosines 627 and 659 is critical for its binding to SHP2, and for activation of the ERK/MAPK pathway in response to EGF.
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