Cell Signaling Technology

Product Pathways - Development

Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb #8814

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IHC-P ChIP H M R Mk (C) (X) (Z) (B) (Dg) (Pg) (GP) (Hr) Endogenous 92 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  C=Chicken  X=Xenopus  Z=Zebrafish  B=Bovine  Dg=Dog  Pg=Pig  GP=Guinea Pig  Hr=Horse
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb recognizes endogenous β-catenin protein when residues Ser33, Ser37, and Thr41 are not phosphorylated. It does not detect β-catenin protein if tri-phosphorylated at Ser33/Ser37/Thr41.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser37 of human β-catenin protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, untreated (-) or treated (+) with LiCl (20 mM, 20 hr at 37ºC), using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb (left) and β-Catenin Antibody #9562 (right). Equal protein loading was confirmed using β-Tubulin (9F3) Rabbit mAb #2128.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, untreated (-) or treated (+) with MG132 (10 μM, 4 hr at 37ºC), using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb (left), Phospho-β-Catenin (Ser33/37/Thr41) Antibody #9561 (center), or β-Catenin (6B3) Rabbit mAb #9582 (right). Note that Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb fails to detect poly-ubiquitinated β-catenin in MG132-treated cells, indicating its specificity for stabilized protein.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin embedded Apc (Min/+) mouse intestinal adenoma using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb.


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin embedded human breast carcinoma using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin embedded cell pellets, HeLa (left) or NCI-H28 (right), using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin embedded mouse colon using Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb in the presence of phospho-β-catenin (Ser33/37/Thr41) peptide (left) or non-phospho-β-catenin (Ser33/37/Thr41) peptide (right). Note the absence of staining only in the presence of the non-phospho-β-catenin (Ser33/37/Thr41) peptide, demonstrating non-phospho specificity.


Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HCT 116 cells and either 5 μl of Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human Axin2 Intron 1 Primers #8973, SimpleChIP® Human CaMK2D Intron 3 Primers #5111, human c-Myc promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. 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.

ELISA-Peptide

ELISA-Peptide

Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb specificity was determined by peptide competition ELISA. Graph depicts binding of the antibody to non-phospho-β-catenin (Ser33/37/Thr41) peptide in the presence of increasing concentrations of competitor peptides. As shown, non-phospho-β-catenin peptides compete for binding, whereas tri-phospho-β-catenin (Ser33/37/Thr41) peptide does not compete for binding.

Background

β-Catenin is a key downstream effector in the Wnt signaling pathway (1). It is implicated in two major biological processes in vertebrates: early embryonic development (2) and tumorigenesis (3). CK1 phosphorylates β-catenin at Ser45. This phosphorylation event primes β-catenin for subsequent phosphorylation by GSK-3β (4-6). GSK-3β destabilizes β-catenin by phosphorylating it at Ser33, Ser37, and Thr41 (7). Mutations at these sites result in the stabilization of β-catenin protein levels and have been found in many tumor cell lines (8).

Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAb is designed to specifically recognize the stabilized form of β-catenin, i.e., protein that has not been phosphorylated by GSK-3, and thus is functionally active in cell-cell adhesion and/or the canonical Wnt signaling pathway.

  1. Cadigan, K.M. and Nusse, R. (1997) Genes Dev. 11, 3286-3305.
  2. Wodarz, A. and Nusse, R. (1998) Annu. Rev. Cell. Dev. Biol. 14, 59-88.
  3. Polakis, P. (1999) Curr. Opin. Genet. Dev. 9, 15-21.
  4. Amit, S. et al. (2002) Genes Dev. 16, 1066-1076.
  5. Lin, C. et al. (2002) Cell 108, 837-847.
  6. Yanagawa, S. et al. (2002) EMBO J. 21, 1733-1742.
  7. Yost, C. et al. (1996) Genes Dev. 10, 1443-1454.
  8. Morin, P.J. (1997) Science 275, 1787-1790.

Application References

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