Cell Signaling Technology

Product Pathways - Autophagy Signaling

ULK1 Antibody Sampler Kit #8359

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
ULK1 (R600) Antibody #4773 40 µl W H Mk 150 Rabbit
Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb #5869 40 µl W IP H M (R) 140 Rabbit IgG
Phospho-ULK1 (Ser757) Antibody #6888 40 µl W H M Mk 140 Rabbit
Raptor (24C12) Rabbit mAb #2280 40 µl W IP H M R Mk 150 Rabbit IgG
AMPKα (D63G4) Rabbit mAb #5832 40 µl W IP H M R Mk B 62 kDa Rabbit
Phospho-AMPKα (Thr172) (40H9) Rabbit mAb #2535 40 µl W IP IHC-P H M R Hm Mk Dm Sc (C) (Z) (B) (Pg) 62 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat
Phospho-Raptor (Ser792) Antibody #2083 40 µl W H M R 150 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  C=Chicken  Dm=D. melanogaster  Z=Zebrafish  B=Bovine  Pg=Pig  Sc=S. cerevisiae
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

ULK1 (R600) Antibody, Raptor (24C12) Rabbit mAb, and AMPKα (D63G4) Rabbit mAb recognize total endogenous levels of the corresponding target proteins irrespective of phosphorylation state. Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb and Phospho-ULK1 (Ser757) Antibody recognize endogenous levels of ULK1 only when phosphorylated at the indicated residues. Phospho-Raptor (Ser792) Antibody recognizes endogenous levels of raptor only when phosphorylated at Ser792. Phospho-AMPKα (Thr172) (40H9) Rabbit mAb recognizes endogenous levels of AMPKα only when phosphorylated at Thr172.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Raptor (24C12) Rabbit mAb #2280.

Western Blotting

Western Blotting

Western blot analysis of extracts from C2C12 cells, untreated (-) or treated (+) with Oligomycin #9996 (0.5 µM), using Phospho-AMPKα (Thr172) (40H9) Rabbit mAb #2535 (upper) or AMPKα Antibody #2532 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from RD and SH-SY5Y cells using ULK1 (R600) Antibody #4773.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using AMPKα (D63G4) Rabbit mAb #5832.

Western Blotting

Western Blotting

Western blot analysis of extracts from MCF7 cells, untreated (-) or treated (+) with Oligomycin #9996 (0.5 μM, 30 min), and C2C12 cells, untreated (-) or treated (+) with hydrogen peroxide (10 mM, 5 min), using Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb #5869.

Western Blotting

Western Blotting

Western blot analysis of extracts from A-431 cells, untreated (-) or treated (+) with Human Epidermal Growth Factor (hEGF) #8916 (100 ng/ml, 30 min) using Phospho-ULK1 (Ser757) Antibody #6888 (upper), or α-Tubulin (11H10) Rabbit mAb #2125 (lower).


Western blot analysis of C2C12 or 293 cells, untreated (-) or treated (+) with AICAR (0.5 mM, 30 min) or Oligomycin #9996 (0.5 μM, 30 min), using Phospho-Raptor (Ser792) Antibody #2083 (upper and lower left) or Raptor (24C12) Rabbit mAb #2280 (upper and lower right). *Cross-reacting bands at 200 kDa.

Description

The ULK1 Antibody Sampler Kit provides an economical way to investigate ULK1 signaling. The kit contains enough primary antibody to perform four western blots with each primary antibody.

Source / Purification

Activation-state specific monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser555 of human ULK1 protein or to residues surrounding Thr172 of human AMPKα protein. Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to human raptor protein or to residues surrounding Lys40 of human AMPKα protein. Activation-state specific polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser757 of human ULK1 protein or to residues surrounding Ser792 of human raptor protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg600 of human ULK1 protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

Two related serine/threonine kinases, UNC-51-like kinase 1 and 2 (ULK1, ULK2), were discovered as mammalian homologs of the C. elegans gene UNC-51 in which mutants exhibited abnormal axonal extension and growth (1-4). Both proteins are widely expressed and contain an amino-terminal kinase domain followed by a central proline/serine rich domain and a highly conserved carboxy-terminal domain. The roles of ULK1 and ULK2 in axon growth have been linked to studies showing that the kinases are localized to neuronal growth cones and are involved in endocytosis of critical growth factors, such as NGF (5). Yeast two-hybrid studies found ULK1/2 associated with modulators of the endocytic pathway, SynGAP and syntenin (6). Structural similarity of ULK1/2 has also been recognized with the yeast autophagy protein Atg1/Apg1 (7). Knockdown experiments using siRNA demonstrated that ULK1 is essential for autophagy (8), a catabolic process for the degradation of bulk cytoplasmic contents (9,10). It appears that Atg1/ULK1 can act as a convergence point for multiple signals that control autophagy (11), and can bind to several autophagy-related (Atg) proteins, regulating phosphorylation states and protein trafficking (12-16).

Raptor mediates the binding of mTORC1 to ULK1, which phosphorylates and inhibits ULK1 under nutrient rich conditions. AMPK also associates directly with ULK1 and, upon nutrient deprivation, can readily reverse the inhibitory effect of mTORC1 by phosphorylating raptor and initiating autophagy (17,18).

  1. Ogura, K. et al. (1994) Genes Dev 8, 2389-400.
  2. Kuroyanagi, H. et al. (1998) Genomics 51, 76-85.
  3. Yan, J. et al. (1998) Biochem Biophys Res Commun 246, 222-7.
  4. Yan, J. et al. (1999) Oncogene 18, 5850-9.
  5. Zhou, X. et al. (2007) Proc Natl Acad Sci USA 104, 5842-7.
  6. Tomoda, T. et al. (2004) Genes Dev 18, 541-58.
  7. Matsuura, A. et al. (1997) Gene 192, 245-50.
  8. Chan, E.Y. et al. (2007) J Biol Chem 282, 25464-74.
  9. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
  10. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ 12 Suppl 2, 1509-18.
  11. Stephan, J.S. and Herman, P.K. (2006) Autophagy 2, 146-8.
  12. Okazaki, N. et al. (2000) Brain Res Mol Brain Res 85, 1-12.
  13. Young, A.R. et al. (2006) J Cell Sci 119, 3888-900.
  14. Kamada, Y. et al. (2000) J Cell Biol 150, 1507-13.
  15. Lee, S.B. et al. (2007) EMBO Rep 8, 360-5.
  16. Hara, T. et al. (2008) J Cell Biol 181, 497-510.
  17. Shang, L. et al. (2011) Proc Natl Acad Sci U S A 108, 4788-93.
  18. Lee, J.W. et al. (2010) PLoS One 5, e15394.

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