Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - PI3K / Akt Signaling

Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb #5482

Applications Reactivity Sensitivity MW (kDa) Isotype
W IHC-P IF-IC F H M R Mk Endogenous 46, 48 Rabbit IgG

Applications Key:  W=Western Blotting  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
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-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb detects endogenous levels of NDRG1 when phosphorylated at Thr346. This antibody likely cross-reacts with other conserved phosporylation sites on NDRG1 at positions Thr356 and Thr366.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr346 of mouse NDRG1 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from C2C12 cells, untreated or treated with LY294002 #9901, insulin, or both using Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb (upper) or total NDRG1 Antibody #5196 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from A431 cells treated with hEGF #8916 (100 ng/ml, 30 minutes), with or without calf intestinal phosphatase and λ-phosphatase, using Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb (upper) or total NDRG1 Antibody #5196 (lower).

Western Blotting

Western Blotting

Western blot analysis of HeLa cells treated with hEGF #8916 (100 ng/ml, 30 minutes), mock transfected or transfected with SignalSilence® NDRG1 siRNA I #6245 or SignalSilence® NDRG1 siRNA II #6257, using Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb (upper) or β-Tubulin (9F3) Rabbit mAb #2128 (lower).


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis on SignalSlide® Phospho-Akt (Ser473) IHC Controls #8101 [paraffin-embedded LNCaP cell pellets, control (left) or LY294002-treated (right)] using NDRG1 Antibody #5196 (top) or Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb (bottom).

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-emebedded mouse colon using Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human cervical carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb.


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-emebedded human lung carcinoma using Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, wortmannin #9951 and U0126 #9903 (blue), using Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of C2C12 cells, treated with LY294002 #9901 (left) or insulin (right), using Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).


Background

N-myc downstream-regulated gene 1 (NDRG1), also termed Cap43, Drg1, RTP/rit42, and Proxy-1, is a member of the NDRG family, which is composed of four members (NDRG1-4) that function in growth, differentiation, and cell survival (1-5). NDRG1 is ubiquitously expressed and highly responsive to a variety of stress signals including DNA damage (4), hypoxia (5), and elevated levels of nickel and calcium (2). Expression of NDRG1 is elevated in N-myc defective mice and is negatively regulated by N- and c-myc (1,6). During DNA damage, NDRG1 is induced in a p53-dependent fashion and is necessary for p53-mediated apoptosis (4,7). Research studies have shown that NDRG1 may also play a role in cancer progression by promoting differentiation, inhibiting growth, and modulating metastasis and angiogenesis (3,4,6,8,9). Nonsense mutation of the NDRG1 gene has been shown to cause hereditary motor and sensory neuropathy-Lom (HMSNL), which is supported by studies demonstrating the role of NDRG1 in maintaining myelin sheaths and axonal survival (10,11). NDRG1 is up-regulated during mast cell maturation and its deletion leads to attenuated allergic responses (12). Both NDRG1 and NDRG2 are substrates of SGK1, although the precise physiological role of SGK1-mediated phosphorylation is not known (13). NDRG1 is phosphorylated by SGK1 at Thr328, Ser330, Thr346, Thr356, and Thr366. Phosphorylation by SGK1 primes NDRG1 for phosphorylation by GSK-3.

Phospho-NDRG1 (Thr346) (D98G11) XP® Rabbit mAb is directed at a site that was identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's LC-MS/MS platform for modification site discovery. Phosphorylation at Thr346 was discovered using an Akt substrate antibody and was shown to be induced by insulin treatment in multiple cell lines. Please visit PhosphoSitePlus®, CST's modification site knowledgebase, at www.phosphosite.org for more information.

  1. Shimono, A. et al. (1999) Mech Dev 83, 39-52.
  2. Zhou, D. et al. (1998) Cancer Res 58, 2182-9.
  3. van Belzen, N. et al. (1997) Lab Invest 77, 85-92.
  4. Kurdistani, S.K. et al. (1998) Cancer Res 58, 4439-44.
  5. Park, H. et al. (2000) Biochem Biophys Res Commun 276, 321-8.
  6. Li, J. and Kretzner, L. (2003) Mol Cell Biochem 250, 91-105.
  7. Stein, S. et al. (2004) J Biol Chem 279, 48930-40.
  8. Maruyama, Y. et al. (2006) Cancer Res 66, 6233-42.
  9. Nishio, S. et al. (2008) Cancer Lett 264, 36-43.
  10. Kalaydjieva, L. et al. (2000) Am J Hum Genet 67, 47-58.
  11. Okuda, T. et al. (2004) Mol Cell Biol 24, 3949-56.
  12. Taketomi, Y. et al. (2007) J Immunol 178, 7042-53.
  13. Murray, J.T. et al. (2004) Biochem J 384, 477-88.

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