Cell Signaling Technology

Product Pathways - MAPK Signaling

PathScan® Phospho-FRA1 (Ser265) Sandwich ELISA Kit #11975

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
Phospho-FRA1(Ser265) Rabbit mAb Coated Microwells 96 tests
FRA1 Rabbit Detection mAb (Biotinylated) 11 ml Green
Streptavidin-HRP 11 ml Red
TMB Substrate #7004 11 ml
STOP Solution #7002 11 ml
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellow

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Phospho-FRA1 (Ser265) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of FRA1 when phosphorylated at Ser265. A Phospho-FRA1 (Ser265) Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, phosphorylated FRA1 protein is captured by the coated antibody. Following extensive washing, a biotinylated FRA1 Rabbit Detection mAb is added to detect the captured phospho-FRA1 (Ser265) protein. HRP-linked streptavidin is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for the developed color is proportional to the quantity of FRA1 phosphorylated at Ser265.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

PathScan® Phospho-FRA1 (Ser265) Sandwich ELISA Kit recognizes endogenous levels of FRA1 protein when phosphorylated at Ser265, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA

ELISA

Figure 2. The relationship between protein concentration of lysates from HeLa cells, untreated and treated with TPA #4174, and the absorbance at 450 nm is shown.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of HeLa cells with TPA stimulates expression and phosphorylation of FRA1 at Ser265, detected by the PathScan® Phospho-FRA1 (Ser265) Sandwich ELISA Kit. HeLa cells (80-90% confluent) were treated with 200 nM TPA #4174 for 4 hr and lysed. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blot using Phospho-FRA1 (Ser265) (D22B1) Rabbit mAb #5841 is shown in the bottom figure.

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), that 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, 921-928.
  2. Dobrzanski, P. et al. (1991) Mol. Cell. Biol. 11, 5470-5478.
  3. Nakabeppu, Y. and Nathans, D. (1991) Cell 64, 751-759.
  4. Rosenberger, S.F. et al. (1999) J. Biol. Chem. 274, 1124-1130.
  5. Sasaki, T. et al. (2006) Mol. Cell 24, 63-75.
  6. Basbous, J. et al. (2007) Mol. Cell. Biol. 27, 3936-3950.
  7. Kovary, K. and Bravo, R. (1991) Mol. Cell. Biol. 11, 2451-2459.
  8. Kovary, K. and Bravo, R. (1992) Mol. Cell. Biol. 12, 5015-5023.

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