Western blot analysis of extracts from 293T cells, untreated (-) or treated with LY294002 #9901 (50 μM, overnight; +) and Wortmannin #9951 (1 μM, overnight; +), using Phospho-FoxO3a (Ser253) (D18H8) Rabbit mAb (upper) or FoxO3a (D19A7) Rabbit mAb #12829 (lower).
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
Phospho-FoxO3a (Ser253) (D18H8) Rabbit mAb detects endogenous levels of FoxO3a only when phosphorylated at Ser253. This antibody may cross-react with FoxO1 when overexpressed and phosphorylated at Ser251 or FoxO4 when overexpressed and phosphorylated at Ser197.
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser253 of human FoxO3a protein.
The Forkhead family of transcription factors is involved in tumorigenesis of rhabdomyosarcoma and acute leukemias (1-3). Within the family, three members (FoxO1, FoxO4, and FoxO3a) have sequence similarity to the nematode orthologue DAF-16, which mediates signaling via a pathway involving IGFR1, PI3K, and Akt (4-6). Active forkhead members act as tumor suppressors by promoting cell cycle arrest and apoptosis. Increased expression of any FoxO member results in the activation of the cell cycle inhibitor p27 Kip1. Forkhead transcription factors also play a part in TGF-β-mediated upregulation of p21 Cip1, a process negatively regulated through PI3K (7). Increased proliferation results when forkhead transcription factors are inactivated through phosphorylation by Akt at Thr24, Ser256, and Ser319, which results in nuclear export and inhibition of transcription factor activity (8). Forkhead transcription factors can also be inhibited by the deacetylase sirtuin (SirT1) (9).
FoxO3a is phosphorylated by Akt at several sites, including Thr32, Ser253 and Ser315. FoxO3a associates with 14-3-3 proteins following Akt phosphorylation and is retained in the cytoplasm. In the absence of survival factors, FoxO3a is dephosphorylated, translocates to the nucleus, and triggers cell death by a Fas ligand-dependent mechanism (7).
Explore pathways + proteins related to this product.