New England Biolabs Canada
 
XP Monoclonal Antibody

Product Pathways - MAPK Signaling

Phospho-c-Fos (Ser32) (D82C12) XP® Rabbit mAb #5348

Item# Description List Price Web Price Qty
5348S Phospho-c-Fos (Ser32) (D82C12) XP® Rabbit mAb - 100 µl $480.00
$432.00
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5348T Phospho-c-Fos (Ser32) (D82C12) XP® Rabbit mAb - 20 µl $192.00
$172.80
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VIEW COMPANION PRODUCTS HIDE COMPANION PRODUCTS
Application Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W Human, Mouse, Rat Endogenous 62 Rabbit IgG
IP
IF-IC
F
ChIP
ChIP-seq

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting, IP=Immunoprecipitation, IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry, ChIP=Chromatin IP, ChIP-seq=Chromatin IP-seq

Homology

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

Directions For Use

For optimal ChIP and ChIP-seq, 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.

Specificity / Sensitivity

Phospho-c-Fos (Ser32) (D82C12) XP® Rabbit mAb detects endogenous levels of c-Fos protein only when phosphorylated on Ser32. The antibody does not cross-react with other Fos proteins, including FosB, FRA1 and FRA2.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to Ser32 of human c-Fos protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, serum-starved overnight and then either untreated or stimulated for 4 hours with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174, using Phospho-c-Fos (Ser32) (D82C12) XP® Rabbit mAb (upper) and c-Fos (9F6) Rabbit mAb #2250 (lower). Antibody phospho-specificity is shown by treating lysates with λ-phosphatase.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, serum-starved (left), TPA-treated (middle) or treated with TPA and λ-phosphatase (right), using Phospho-c-Fos (Ser32) (D82C12) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HeLa cells, untreated (blue) or TPA-treated (green), using Phospho-c-Fos (Ser32) (D82C12) XP® Rabbit mAb.


Chromatin IP-seq

Chromatin IP-seq

Chromatin immunoprecipitations were performed with cross-linked chromatin from PC-12 cells starved overnight and treated with Human β-Nerve Gowth Factor (hβ-NGF) #5221 (50ng/ml) for 2h and Phospho-c-Fos (Ser32) (D82C12) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across Fbxl19 gene. For additional ChIP-seq tracks, please download the product data sheet.

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from PC-12 cells starved overnight and treated with Human β-Nerve Gowth Factor (hβ-NGF) #5221 (50ng/ml) for 2h and either Phospho-c-Fos (Ser32) (D82C12) XP® 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 using 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

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!


 

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