Peptide Competition – Mutant Analysis Lysates prepared from COS cells overexpressing human wild-type PAG3 alone (1) cotransfected with human wild-type PAG3 and mouse Pyk2 (2-5), or mutant [Y724F/Y763F] PAG3 and mouse Pyk2 (6) were resolved by SDS-PAGE on an 8% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with PAG3 [pY724] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2, 6), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphotyrosine-containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab and quote;)2 anti-rabbit IgG HRP conjugate (Cat. no. ALI4404) in 3% BSA-TBST buffer, and bands were detected using the Pierce SuperSignal™ method. The data show that only the peptide corresponding to PAG3 [pY724] blocks the signal, and the signal is absent in the mutant extracts, demonstrating the specificity of the antibody. Cell extracts were kindly provided by Dr. Hisataka Sabe, Osaka Bioscience Institute, Osaka, Japan.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human PAG3 that contains tyrosine 724.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:1,000|
* 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.
ASAP2 is a multidomain protein belonging to the beta family with two ANK repeats, an Arf-GAP domain, a PH domain and a SH3 domain. This protein localizes in the Golgi apparatus and also at the plasma membrane where it colocalizes with protein tyrosine kinase 2-beta (PYK2) and forms a stable complex with PYK2 in vivo. ASAP2 is tyrosine phosphorylated by activated PYK2. It functions as a substrate and downstream target for PYK2 and SRC, a pathway that may be involved in the regulation of vesicular transport. ASAP2 activates the small GTPases ARF1, ARF5 and ARF6 and regulates the formation of post-Golgi vesicles and modulates constitutive secretion. It modulates phagocytosis mediated by Fc gamma receptor and ARF6. Usually detected in heart, brain, placenta, kidney, monocytes and pancreas, it is up-regulated during monocyte maturation. Multiple transcript variants encoding different isoforms have been found.
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