Stimulated Emission Depletion (STED) Nanoscopy of Akt-phospho-Ser473
Akt is a component of the PI-3 kinase pathway and is activated by phosphorylation at Ser 473 and Thr 308.
Akt is a cytoplasmic protein also known as AKT1, Protein Kinase B (PKB) and rac (related to A and C kinases). Akt is a key regulator of many signal transduction pathways. Akt exhibits tight control over cell proliferation and cell viability. Overexpression or inappropriate activation of Akt is noted in many types of cancer. Akt mediates many of the downstream events of PI 3-kinase (a lipid kinase activated by growth factors, cytokines and insulin). PI 3-kinase recruits Akt to the membrane, where it is activated by PDK1 phosphorylation. Once phosphorylated, Akt dissociates from the membrane and phosphorylates targets in the cytoplasm and the cell nucleus. AKT has two main roles: (i) inhibition of apoptosis; (ii) promotion of proliferation.
Phospho PI3K/AKT Signaling is Colocalized with Microtubules in High Resolution STED Nanoscopy Images
Data was collected using Leica Microsystems
high resolution TCS STED imaging system. Rockland Immunochemicals Anti-phospho-Akt (Ser473) Monoclonal Antibody
is functional by western blotting, immunohistochemistry, and immunofluorescence microscopy
High resolution STED IF nanoscopy is shown above. Rockland Immunochemicals Anti-phospho-Akt (Ser473) monoclonal antibody
(Cat no: 200-301-268) was used
at 10 ug/ml for 1 h at RT to stain A431 cells after serum deprivation for 12h.
Cells were fixed in 4% PFA for 5 and after washes blocked with 10% NGS / 0.2% Triton X-100 for 30. Phosphorylated Akt (red) is observed to be localized in the cytoplams (A) and also organised at the periphery of the cells (D). Sequential staining at 1.4 ug/ml for 1 h at RT with Anti-alpha-Tubulin
(Rabbit) (Cat no: 600-401-880) is shown in panels B & E. The merge images (C & F) show phospho-Akt (Ser473) colocalized with the microtubule network. High magnification images (D, E & F) show phospho-AKT (Ser473) appears to be proximal to microtubules.
Parallel experiments using Anti-Akt Monoclonal Antibody
(Cat no: 200-301-401) showed no colocalization (not shown).
Images ware taken with a 100X objective on a Leica TCS STED (Stimulated Emission a Depletion), Leica Microsystems, Inc. Exton, PA, USA.
Personal communication, Myriam Gastard, Leica Microsystems, Inc. Exton, PA, USA.
See also: Akt Signaling Antibodies by Rockland Immunochemicals
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