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Western Blot (Cell lysate)

Western blot using AKT1 (phospho S473) monoclonal antibody, clone 17F6.B11 (Cat # MAB1919) shows detection of phospho-AKT1 (indicated by arrowhead at ~56 KDa) on PDGF stimulated NIH/3T3 cell lysates (Lane 2).
No reactivity is seen for non-phosphorylated AKT1 in untreated cells (Lane 1).
Each lane contained approximately 10 ug of lysate.
All samples were loaded on to a 4-20% gradient gel for separation.
After electrophoresis, the gel was blocked with 5% BLOTTO in TBS for 90 min at RT.
The membrane was probed with the primary antibody at a 1:10,000 dilution in TBS with 0.05% Tween-20 with 1% BSA, for 1 h at 4°C. For detection HRP conjugated Gt-a-Mouse IgG was used at a 1:20,000 dilution for 1 h at 4°C with FemtoMax™ enhanced chemiluminescent reagent.
Images were captured using 2X2 binning for 10-20 sec using a BioSpectrum Imaging System (UVP Ltd.).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)

Immunohistochemistry of formalin fixed and paraffin embedded using AKT1 (phospho S473) monoclonal antibody, clone 17F6.B11 (Cat # MAB1919) shows detection of Phospho-AKT1 S473 in human prostate tissue.
The antibody was used at 20 ug/mL .
The staining is much stronger than the weak basal level of phosphorylation in normal prostate tissue.
Signal was developed using Dako's Techmate streptavidin-biotin reagents.
Personal communication, Glenna Burmer, Lifespan Biosciences, Seattle, WA.

Enzyme-linked Immunoabsorbent Assay

Entrez GeneID

207

Protein Accession#

P31749;AAH00479

Gene Name

AKT1

Gene Alias

AKT, MGC99656, PKB, PKB-ALPHA, PRKBA, RAC, RAC-ALPHA

Gene Description

v-akt murine thymoma viral oncogene homolog 1

Omim ID

164730 181500

Gene Ontology

Hyperlink

Gene Summary

The serine-threonine protein kinase encoded by the AKT1 gene is catalytically inactive in serum-starved primary and immortalized fibroblasts. AKT1 and the related AKT2 are activated by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKT1. It was shown that the activation occurs through phosphatidylinositol 3-kinase. In the developing nervous system AKT is a critical mediator of growth factor-induced neuronal survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKT1, which then phosphorylates and inactivates components of the apoptotic machinery. Multiple alternatively spliced transcript variants have been found for this gene. [provided by RefSeq

Other Designations

RAC-alpha serine/threonine-protein kinase|murine thymoma viral (v-akt) oncogene homolog-1|protein kinase B|rac protein kinase alpha