Western blot analysis of extracts from untreated or serum-treated COS-7 cells exogenously expressing GFP-FoxO1, using Phospho-FoxO1 (Ser319) Antibody. The phospho-specificity of the antibody was verified by treating the membrane with (+) or without (-) calf intestinal phosphatase (CIP) after Western transfer.
Western blot analysis of extracts from serum-treated COS-7 cells untransfected, exogenously expressing FoxO4 or GFP-FoxO1, using Phospho-FoxO1 (Ser319) (left) or Phospho-FoxO1 (Ser319)/FoxO4 (Ser262) Antibody #2487 (right).
|MW (kDa)||120 (GFP-FoxO1)|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: 10
Phospho-FoxO1 (Ser319) Antibody detects exogenous levels of FoxO1 only when phosphorylated at serine 319. The antibody does not cross-reacts with FoxO1 phosphorylated at other sites nor with other phosphorylated family members.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser319 of human FoxO1. Antibodies are purified by protein A and peptide affinity chromatography.
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).
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