Product Pathways - Akt Signaling

Forkhead Signaling Antibody Sampler Kit #9946

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Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey

Specificity / Sensitivity

Phospho-FoxO1 (Thr24)/FoxO3a (Thr32) Antibody detects endogenous levels of FoxO1/FoxO3a only when phosphorylated at Thr24 of FoxO1 or Thr32 of FoxO3a. The antibody cross-reacts with phosphorylated FoxO4 at Thr28, but not with FoxO1 family members phosphorylated at other sites. Phospho-FoxO1 (Ser256) Antibody detects endogenous levels of FoxO1 only when phosphorylated at Ser256. The antibody cross-reacts with Fox04 phosphorylated at Ser193. Phospho-FoxO1 (Ser319)/FoxO4 (Ser262) Antibody detects transfected levels of FoxO1 and FoxO4 only when phosphorylated at Ser319 and Ser262, respectively. The antibody does not cross-react with FoxO1 or FoxO4 phosphorylated at other sites, nor with phosphorylted FoxO3a. FoxO1 Antibody detects endogenous levels of FoxO1 protein and FoxO4 (at 65-70 kDa). The antibody is sensitive to phosphorylation within the antigenic site and will preferrentially detect FoxO1 (or FoxO4) when not phosphorylated. Phospho-Fox03a (Ser253) Antibody detects endogenous levels of FoxO3a only when phosphorylated at Ser253. The antibody does not detect the transfected levels of FoxO4 or FoxO1. The antibody reacts with denatured components of bovine serum including BSA. Phospho-FoxO3a (Ser318/321) Antibody detects endogenous levels of FoxO3a only when phosphorylated at Ser318/321. The antibody is expected to cross-react with FoxO1 when phosphorylated at Ser322/325 based on the peptide sequence. FoxO3a Antibody detect endogenous levels of total FoxO3a protein. The antibody does not detect transfected levels of FoxO4 or FoxO1. The antibody reacts with denatured components of bovine serum including BSA. The FoxO4 Antibody detects endogenous levels of FoxO4. The antibody is sensitive to phosphorylation within the antigen and preferrentially detects unphosphorylated FoxO4.

Western Blotting

Western blot analysis of extracts from serum-treated COS-7 cells transfected with FoxO4 or FoxO3a using Phospho-FoxO1 (Ser319)/FoxO4 (Ser262) Antibody #2487. The phospho-specificity of the antibody was verified by treating the membrane with (+) or without (-) calf intestinal phosphatase (CIP) after Western transfer.

Western Blotting

Western blot analysis of extracts from COS cells, serum starved or serum treated, using Phospho-FoxO1 (Ser256) Antibody #9461.

Western Blotting

Western blot analysis of extracts from COS cells, untreated or serum-treated (20%), using FoxO1 Antibody #9462.

Western Blotting

Western blot analysis of extracts from HT29 cells, serum starved or serum treated, using Phospho-FoxO1 (Thr24)/FoxO3a (Thr32) Antibody #9464.

Western Blotting

Western blot analysis of extracts from serum starved and serum-treated NIH/3T3 cells and untreated and LY294002/Wortmannin-treated MDA-MB-468 cells using Phospho-FoxO3a (Ser318/321) Antibody #9465 (upper) or Akt Antibody #9272 (lower).

Western Blotting

Western blot analysis of extracts from PC3 cells, untreated or treated with λ phosphatase, using Phospho-FoxO3a (Ser253) Antibody #9466 (upper) or Akt Antibody #9272 (lower).

Western Blotting

Western blot analysis of extracts from COS cells, mock transfected (lane 1), transfected with FoxO4 (lane 2), transfected with FoxO1 (lane 3), or transfected with FoxO3a (lane 4), using FoxO3a Antibody #9467.

Western Blotting

Western blot analysis of extracts from Ramos and Jurkat cells using FoxO4 Antibody #9472.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with a synthetic peptide (KLH-coupled) derived from the sequence of human FoxO1, FoxO3a or FoxO4. Antibodies are purified by protein A and peptide affinity chromatography. Antibodies are supplied in HEPES buffer with 50% glycerol and less than 0.02% sodium azide.

Background

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 p27Kip1. Forkhead transcription factors also play a part in TGF-β-mediated upregulation of p21CIP1, 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).

1. Anderson, M.J. et al. (1998) Genomics 47, 187-199.
2. Galili, N. et al. (1993) Nat. Genet. 5, 230-235.
3. Borkhardt, A. et al. (1997) Oncogene 14, 195-202.
4. Nakae, J. et al. (1999) J. Biol. Chem. 274, 15982-15985.
5. Rena, G. et al. (1999) J. Biol. Chem. 274, 17179-17183.
6. Guo, S. et al. (1999) J. Biol. Chem. 274, 17184-17192.
7. Seoane, J. et al. (2004) Cell 117, 211-223.
8. Arden, K.C. (2004) Mol. Cell 14, 416-418.
9. Yang, Y. et al. (2005) EMBO J. 24, 1021-1032.

Application References

• Rena, G. et al. (2002) EMBO J 21, 2263-71. This article references the use of Forkhead Signaling Antibody Sampler Kit in the following applications: Western Blotting
• Richards, J.S. et al. (2002) Mol Endocrinol 16, 580-99. This article references the use of Forkhead Signaling Antibody Sampler Kit in the following applications: Western Blotting
• Stahl, M. et al. (2002) J Immunol 168, 5024-31. This article references the use of Forkhead Signaling Antibody Sampler Kit in the following applications: Western Blotting
• Fan, S. et al. (2001) Mol Cell Biol 21, 4968-84. This article references the use of Forkhead Signaling Antibody Sampler Kit in the following applications: Western Blotting
• Iordanov, M.S. et al. (2002) Mol Cell Biol 22, 5380-94. This article references the use of Forkhead Signaling Antibody Sampler Kit in the following applications: Western Blotting

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