Revision 1
#89267
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For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:
W, IP
Reactivity:
H M R
Sensitivity:
Endogenous
MW (kDa):
140
Source/Isotype:
Rabbit IgG
UniProt ID:
#O75385
Entrez-Gene Id:
8408
Product Usage Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:200 |
Storage
Specificity/Sensitivity
Phospho-ULK1 (Ser317) (F1Z6C) Rabbit mAb recognizes endogenous levels of ULK1 protein only when phosphorylated at Ser317.
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser317 of human ULK1 protein.
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).
AMPK, activated during low nutrient conditions, directly phosphorylates ULK1 at multiple sites, including Ser317, Ser555, and Ser777 (17,18). Conversely, mTOR, which is a regulator of cell growth and an inhibitor of autophagy, phosphorylates ULK1 at Ser757 and disrupts the interaction between ULK1 and AMPK (17).
Background References
- Ogura, K. et al. (1994) Genes Dev 8, 2389-400.
- Kuroyanagi, H. et al. (1998) Genomics 51, 76-85.
- Yan, J. et al. (1998) Biochem Biophys Res Commun 246, 222-7.
- Yan, J. et al. (1999) Oncogene 18, 5850-9.
- Zhou, X. et al. (2007) Proc Natl Acad Sci USA 104, 5842-7.
- Tomoda, T. et al. (2004) Genes Dev 18, 541-58.
- Matsuura, A. et al. (1997) Gene 192, 245-50.
- Chan, E.Y. et al. (2007) J Biol Chem 282, 25464-74.
- Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
- Codogno, P. and Meijer, A.J. (2005) Cell Death Differ 12 Suppl 2, 1509-18.
- Stephan, J.S. and Herman, P.K. (2006) Autophagy 2, 146-8.
- Okazaki, N. et al. (2000) Brain Res Mol Brain Res 85, 1-12.
- Young, A.R. et al. (2006) J Cell Sci 119, 3888-900.
- Kamada, Y. et al. (2000) J Cell Biol 150, 1507-13.
- Lee, S.B. et al. (2007) EMBO Rep 8, 360-5.
- Hara, T. et al. (2008) J Cell Biol 181, 497-510.
- Kim, J. et al. (2011) Nat Cell Biol 13, 132-41.
- Egan, D.F. et al. (2011) Science 331, 456-61.
Species Reactivity
Species reactivity is determined by testing in at least one approved application (e.g., western blot).
Western Blot Buffer
IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
Applications Key
W: Western Blotting IP: Immunoprecipitation
Cross-Reactivity Key
H: Human M: Mouse R: Rat Hm: Hamster Mk: Monkey Vir: Virus Mi: Mink C: Chicken Dm: D. melanogaster X: Xenopus Z: Zebrafish B: Bovine Dg: Dog Pg: Pig Sc: S. cerevisiae Ce: C. elegans Hr: Horse GP: Guinea Pig Rab: Rabbit G: Goat All: All Species Expected
Trademarks and Patents
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Revision 1
Western blot analysis of extracts from PANC-1 cells, untreated (-) or treated with AMPK activator Compound 991 (50 μM, 1 hr; +), using Phospho-ULK1 (Ser317) (F1Z6C) Rabbit mAb (upper), ULK1 (D8H5) Rabbit mAb #8054 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western blot analysis of extracts from PANC-1 cells treated with the AMPK activator Compound 991 (50 μM, 1 hr) that were subsequently untreated (-) or treated (+) with λ phosphatase and calf intestinal phosphatase (CIP), using Phospho-ULK1 (Ser317) (F1Z6C) Rabbit mAb (upper), ULK1 (D8H5) Rabbit mAb #8054 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Western blot analysis of extracts from control U-2 OS cells or CRISPR/Cas9 ULK1 knockout (KO) U-2 OS cells, untreated (-) or treated with AMPK activator Compound 991 (50 μM, 1 hr; +), using Phospho-ULK1 (Ser317) (F1Z6C) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in ULK1 KO cells confirms the specificity of the antibody for ULK1. ULK1 KO cells were kindly provided by Dr. Reuben Shaw, Salk Institute for Biological Studies, La Jolla, CA.
Revision 1
Western blot analysis of extracts from various cell lines using Phospho-ULK1 (Ser317) (F1Z6C) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Immunoprecipitation of phospho-ULK1 (Ser317) protein from PANC-1 cell extracts. Cells were treated with AMPK activator Compound 991 (50 μM, 1 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-ULK1 (Ser317) (F1Z6C) Rabbit mAb. Western blot analysis was performed using Phospho-ULK1 (Ser317) (F1Z6C) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.