Cell Signaling Technology

Product Pathways - Development

Phospho-Numb (Ser276) (D5C2) Rabbit mAb #9878

Applications Reactivity Sensitivity MW (kDa) Isotype
W IF-IC H (M) (R) (C) (X) (Z) (B) (Hr) Endogenous 78 Rabbit IgG

Applications Key:  W=Western Blotting  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  M=Mouse  R=Rat  C=Chicken  X=Xenopus  Z=Zebrafish  B=Bovine  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

Phospho-Numb (Ser276) (D5C2) Rabbit mAb recognizes endogenous levels of numb protein only when phosphorylated at Ser276.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser276 of human numb protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa and U-87 MG cells, untreated or treated with either λ phosphatase or TPA #4174 (200 nM, 30 min), using Phospho-Numb (Ser276) (D5C2) Rabbit mAb (upper) or Numb (C29G11) Rabbit mAb #2756 (lower).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with either TPA #4174 (center) or TPA and λ phosphatase (right), using Phospho-Numb (Ser276) (D5C2) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Background

Numb contains an amino-terminal phosphotyrosine-binding (PTB) domain and carboxy-terminal endocytic binding motifs for α-adaptin and EH (Eps15 homology) domain-containing proteins, indicating a role in endocytosis (1,2). There are four mammalian Numb splicing isoforms that are differentially expressed and may have distinct functions (3-5). Numb acts as a negative regulator of Notch signaling by promoting ubiquitination and degradation of Notch (6). The protein is asymmetrically segregated into one daughter cell during cell division, producing two daughter cells with different responses to Notch signaling and different cell fates (7,8). The localization of Numb can also be regulated by G-protein coupled receptor (GPCR) and PKC signaling (9).

Numb can be phosphorylated at several sites including Ser7, Ser276, and Ser295. Phosphorylation at these sites regulates asymmetric membrane localization of Numb and integrin endocytosis (10-12).

  1. Berdnik, D. et al. (2002) Dev. Cell 3, 221-231.
  2. Santolini, E. et al. (2000) J. Cell Biol. 151, 1345-1352.
  3. Dho, S.E. et al. (1999) J. Biol. Chem. 274, 33097-33104.
  4. Verdi, J.M. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 10472-10476.
  5. Toriya, M. et al. (2006) Dev. Neurosci. 28, 142-155.
  6. McGill, M.A. and McGlade, C.J. (2003) J. Biol. Chem. 278, 23196-23203.
  7. Verdi, J.M. et al. (1996) Curr. Biol. 6, 1134-1145.
  8. Reugels, A.M. et al. (2006) Dev. Dyn. 235, 934-948.
  9. Dho, S.E. et al. (2006) Mol. Biol. Cell 17, 4142-4155.
  10. Nishimura, T. and Kaibuchi, K. (2007) Dev Cell 13, 15-28.
  11. Smith, C.A. et al. (2007) EMBO J 26, 468-80.
  12. Wirtz-Peitz, F. et al. (2008) Cell 135, 161-73.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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