Peptide Competition and Mutant Analysis. Lysates prepared from HEK293 cells transiently transfected with WT PTEN (1, 3-6) or mutant S385A (2) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-5) or treated with lambda phosphatase (6), blocked with a 3% Milk-TBST buffer for one hour at room temperature, and incubated with PTEN [pS385] antibody for two hours at room temperature in a 3% Milk-TBST buffer, following prior incubation with: no peptide (1, 2, 6), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-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) and bands were detected using the Pierce SuperSignal™ method.The data show that only the peptide corresponding to PTEN [pS385] blocks the antibody signal. Moreover, the signal is lost in the S385A single mutant (kindly provided by Dr. A. Hall). The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.
|Tested species reactivity||Human|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human PTEN that contains serine 385. The sequence is conserved in mouse and rat.|
|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.
|Western Blot (WB)||See 1 publications below|
This gene was identified as a tumor suppressor that is mutated in a large number of cancers at high frequency. The protein encoded this gene is a phosphatidylinositol-3, 4, 5-trisphosphate 3-phosphatase. It contains a tensin like domain as well as a catalytic domain similar to that of the dual specificity protein tyrosine phosphatases. Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates. It negatively regulates intracellular levels of phosphatidylinositol-3, 4, 5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating AKT/PKB signaling pathway.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Loss of NDRG2 expression activates PI3K-AKT signalling via PTEN phosphorylation in ATLL and other cancers.
44-1064G was used in western blot to present a mechanism for the N-myc downstream-regulated gene 2-dependent regulation of PTEN phosphatase activity via the dephosphorylation of PTEN.
|Nakahata S,Ichikawa T,Maneesaay P,Saito Y,Nagai K,Tamura T,Manachai N,Yamakawa N,Hamasaki M,Kitabayashi I,Arai Y,Kanai Y,Taki T,Abe T,Kiyonari H,Shimoda K,Ohshima K,Horii A,Shima H,Taniwaki M,Yamaguchi R,Morishita K||Nature communications (5:null)||2014|