Western blot analysis of extracts from Jurkat cells treated with either Calyculin A (#9902) or LY294002 (#9901), NIH3T3 and COS-7 cells using Phospho-FoxO1 (Thr24)/(FoxO3a (Thr32)/FoxO4 (Thr28) (4G6) Rabbit mAb to detect FoxO1, FoxO3a and FoxO4 when phosphorylated at the Thr24, Thr32, and Thr28 positions, respectively (left panel). Total FoxO1, FoxO3a and FoxO4 were detected using FoxO1 (C29H4) Rabbit mAb (#2880), FoxO3a (75D8) Rabbit mAb (#2497) and FoxO4 Antibody (#9472), respectively (right panel).
Western blot analysis of extracts from Jurkat cells treated with either Calyculin A (#9902) or LY294002 (#9901) using Phospho-FoxO1 (Thr24)/(FoxO3a (Thr32)/FoxO4 (Thr28) (4G6) Rabbit mAb. The phospho-specificity of the antibody was verified by treating the membrane in the absence (-) or presence (-) of calf intestinal phosphatase (CIP) after western transfer.
|REACTIVITY||H M Mk|
|MW (kDa)||65, 78 to 82, 95|
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 BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Phospho-FoxO1 (Thr24)/FoxO3a (Thr32)/Fox04 (Thr28) (4G6) Rabbit mAb detects endogenous levels of FoxO1 when phosphorylated at Thr24, of FoxO3a when phosphorylated at Thr32 or FoxO4 when phosphorylated at Thr28.Species Reactivity:
Human, Mouse, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr28 of human Fox04.
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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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.