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Product Pathways - MAPK Signaling

Phospho-FRA1 (Ser265) (D22B1) Rabbit mAb #5841

Item# Description List Price Web Price Qty
5841S Phospho-FRA1 (Ser265) (D22B1) Rabbit mAb - 100 µl $433.00
$389.70
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5841T Phospho-FRA1 (Ser265) (D22B1) Rabbit mAb - 20 µl $174.00
$156.60
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VIEW COMPANION PRODUCTS HIDE COMPANION PRODUCTS
Application Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W Human, Mouse, Rat Endogenous 40 Rabbit IgG
ChIP

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting, ChIP=Chromatin IP

Homology

Species predicted to react based on 100% sequence homology: Monkey, Bovine, Horse.

Directions For Use

For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

Protocols

Specificity / Sensitivity

Phospho-FRA1 (Ser265) (D22B1) Rabbit mAb recognizes endogenous levels of FRA1 protein only when phosphorylated at Ser265. This antibody may also cross-react with phospho-FRA2, but does not cross-react with phospho-c-Fos or phospho-FosB.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser265 of human FRA1 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, serum-starved overnight, and either left untreated or treated with TPA #4174 for 4 hours, using Phospho-FRA1 (Ser265) (D22B1) Rabbit mAb #5841 (upper) and FRA1 (D80B4) Rabbit mAb #5281 (lower). Antibody phospho-specificity is shown by treating lysates with λ phosphatase.

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from PC-12 cells starved overnight and treated with β-NGF #5221 (50ng/ml) for 2h and either Phospho-FRA1 (Ser265) (D22B1) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR SimpleChIP® Rat CCRN4L Promoter Primers #7983, rat DCLK1 promoter primers, and SimpleChIP® Rat GAPDH Promoter Primers #7964. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Background

The Fos family of nuclear oncogenes includes c-Fos, FosB, Fos-related antigen 1 (FRA1), and Fos-related antigen 2 (FRA2) (1). While most Fos proteins exist as a single isoform, the FosB protein exists as two isoforms: full-length FosB and a shorter form, FosB2 (Delta FosB), which lacks the carboxy-terminal 101 amino acids (1-3). The expression of Fos proteins is rapidly and transiently induced by a variety of extracellular stimuli including growth factors, cytokines, neurotransmitters, polypeptide hormones, and stress. Fos proteins dimerize with Jun proteins (c-Jun, JunB, and JunD) to form Activator Protein-1 (AP-1), a transcription factor that binds to TRE/AP-1 elements and activates transcription. Fos and Jun proteins contain the leucine-zipper motif that mediates dimerization and an adjacent basic domain that binds to DNA. The various Fos/Jun heterodimers differ in their ability to transactivate AP-1 dependent genes. In addition to increased expression, phosphorylation of Fos proteins by Erk kinases in response to extracellular stimuli may further increase transcriptional activity (4-6). Phosphorylation of c-Fos at Ser32 and Thr232 by Erk5 increases protein stability and nuclear localization (5). Phosphorylation of FRA1 at Ser252 and Ser265 by Erk1/2 increases protein stability and leads to overexpression of FRA1 in cancer cells (6). Following growth factor stimulation, expression of FosB and c-Fos in quiescent fibroblasts is immediate, but very short-lived, with protein levels dissipating after several hours (7). FRA1 and FRA2 expression persists longer, and appreciable levels can be detected in asynchronously growing cells (8). Deregulated expression of c-Fos, FosB, or FRA2 can result in neoplastic cellular transformation; however, Delta FosB lacks the ability to transform cells (2,3).

  1. Tulchinsky, E. (2000) Histol Histopathol 15, 921-8.
  2. Dobrazanski, P. et al. (1991) Mol Cell Biol 11, 5470-8.
  3. Nakabeppu, Y. and Nathans, D. (1991) Cell 64, 751-9.
  4. Rosenberger, S.F. et al. (1999) J Biol Chem 274, 1124-30.
  5. Sasaki, T. et al. (2006) Mol Cell 24, 63-75.
  6. Basbous, J. et al. (2007) Mol Cell Biol 27, 3936-50.
  7. Kovary, K. and Bravo, R. (1991) Mol Cell Biol 11, 2451-9.
  8. Kovary, K. and Bravo, R. (1992) Mol Cell Biol 12, 5015-23.

Application References

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