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8359
ULK1 Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit
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ULK1 Antibody Sampler Kit #8359

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ULK1 Antibody Sampler Kit: Image 1
Immunoprecipitation of ULK1 from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is ULK1 (D8H5) Rabbit mAb, #8054. Western blot was performed using ULK1 (D8H5) Rabbit mAb.
ULK1 Antibody Sampler Kit: Image 2
Simple Western™ analysis of lysates (0.1 mg/mL) from RD cells using ULK1 (D8H5) Rabbit mAb #8054. The virtual lane view (left) shows a single target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
ULK1 Antibody Sampler Kit: Image 3
Western blot analysis of C2C12 or 293 cells, untreated or treated with AICAR (0.5 mM for 30 minutes) or oligomycin (0.5 μM for 30 minutes), using Phospho-Raptor (Ser792) Antibody (upper and lower left ) or Raptor Antibody #2280 (upper and lower right).

*Cross-reacting bands at 200 kDa.

ULK1 Antibody Sampler Kit: Image 4
Western blot analysis of extracts from various cell lines, using Raptor (24C12) Rabbit mAb.
ULK1 Antibody Sampler Kit: Image 5
Western blot analysis of extracts from C2C12 cells, untreated or oligomycin-treated (0.5 µM), using Phospho-AMPKα (Thr172) (40H9) Rabbit mAb (upper) or AMPKα Antibody #2532 (lower).
ULK1 Antibody Sampler Kit: Image 6
Western blot analysis of extracts from HeLa, K-562, C6, and Neuro-2a cells using AMPKα (D63G4) Rabbit mAb.
ULK1 Antibody Sampler Kit: Image 7
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 991 (50 μM, 1 hr; +) using Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb (upper), ULK1 (D8H5) Rabbit mAb #8054 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in ULK1 KO cells confirms the specificity of the antibodies for ULK1. ULK1 knockout cells were kindly provided by Dr. Reuben Shaw, Salk Institute for Biological Studies, La Jolla, CA.
ULK1 Antibody Sampler Kit: Image 8
Western blot analysis of extracts from A172 cells, untreated (-), Torin 1-treated (+; 250 nM; 5 hrs), or INK-128-treated (+; 250 nM; 5 hrs) using Phospho-ULK1 (Ser757) Antibody (upper) or total ULK1 (D8H5) Rabbit mAb #8054 (lower).
ULK1 Antibody Sampler Kit: Image 9
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
ULK1 Antibody Sampler Kit: Image 10
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 991 (50 μM, 1 hr; +) using ULK1 (D8H5) Rabbit mAb (upper), Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb #5869 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in ULK1 KO cells confirms the specificity of the antibodies for ULK1.ULK1 knockout cells were kindly provided by Dr. Reuben Shaw, Salk Institute for Biological Studies, La Jolla, CA.
ULK1 Antibody Sampler Kit: Image 11
Western blot analysis of wild-type (WT) and AMPKα1 and α2 knockout (KO) mouse embryonic fibroblasts (MEFs), untreated or treated with AICAR (2 mM for 1 hour), using Phospho-Raptor (Ser792) Antibody (upper) or Raptor Antibody #4978 (lower). (Image provided by Dr. Reuben Shaw, Salk Institute for Biological Studies).

*Cross-reacting bands at 60, 70 and 240 kDa

ULK1 Antibody Sampler Kit: Image 12
Western blot analysis of extracts from MCF7 cells, untreated or treated with oligomycin #9996 (0.5 μM, 30 minutes), and C2C12 cells, untreated or treated with hydrogen peroxide (10 mM, 5 minutes), using Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb.
ULK1 Antibody Sampler Kit: Image 13
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 (upper), or α-Tubulin (11H10) Rabbit mAb #2125 (lower).
ULK1 Antibody Sampler Kit: Image 14
Western blot analysis of extracts from various cell lines using ULK1 (D8H5) Rabbit mAb.
ULK1 Antibody Sampler Kit: Image 15
Immunohistochemical analysis of paraffin-embedded NCI-H228 cell pellets, control (left) or phenformin-treated (right), using Phospho-AMPKalpha (T172) (40H9) Rabbit mAb.
ULK1 Antibody Sampler Kit: Image 16
Western blot analysis of extracts from MCF7 cells, untreated or treated with oligomycin #9996 (0.5 µM, 30 minutes), using Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb (left). Phospho-specificity is demonstrated by pre-incubating the antibody with phosphorylated (middle) or non-phosphorylated peptides (right) against a region surrounding Ser555 of ULK1.
ULK1 Antibody Sampler Kit: Image 17
Western blot analysis of extracts from wild-type MEF and ULK1 (-/-) MEF cells using ULK1 (D8H5) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). MEF cells were kindly provided by Dr. Reuben Shaw (Salk Institute, La Jolla, CA).
ULK1 Antibody Sampler Kit: Image 18
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-AMPKα (Thr172) (40H9) Rabbit mAb.
ULK1 Antibody Sampler Kit: Image 19
Western blot analysis of extracts from MCF7 cells, untreated (-) or treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (100 μM, 2 hr; +), using Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb (upper) or ULK1 (D8H5) Rabbit mAb #8054 (lower).
ULK1 Antibody Sampler Kit: Image 20
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-AMPKα (Thr172) (40H9) Rabbit mAb.
ULK1 Antibody Sampler Kit: Image 21
Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Phospho-AMPKα (Thr172) (40H9) Rabbit mAb.
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To Purchase # 8359
製品# サイズ 数量 価格 在庫
8359T		 1 Kit  (7 x 20 microliters)

Custom Formulation

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-AMPKα (Thr172) (40H9) Rabbit mAb 2535 20 µl
  • WB
  • IP
  • IHC
 H M R Hm Mk Dm Sc 62 Rabbit IgG
AMPKα (D63G4) Rabbit mAb 5832 20 µl
  • WB
  • IP
 H M R Mk B 62 Rabbit 
Phospho-Raptor (Ser792) Antibody 2083 20 µl
  • WB
 H M R 150 Rabbit 
Raptor (24C12) Rabbit mAb 2280 20 µl
  • WB
  • IP
 H M R Mk 150 Rabbit 
Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb 5869 20 µl
  • WB
  • IP
 H M 140-150 Rabbit IgG
Phospho-ULK1 (Ser757) Antibody 6888 20 µl
  • WB
 H M Mk 140-150 Rabbit 
ULK1 (D8H5) Rabbit mAb 8054 20 µl
  • WB
  • IP
 H M R Mk 150 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
 Rab Goat 

Product Description

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

Specificity / Sensitivity

ULK1 (D8H5) Rabbit mAb, 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.

Source / Purification

Activation-state specific monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptides corresponding to residues surrounding Ser555 of human ULK1 protein or residues surrounding Thr172 of human AMPKα protein. Monoclonal antibodies are produced by immunizing animals with a synthetic peptides corresponding to human raptor protein, residues surrounding Lys40 of human AMPKα protein, or residues surrounding Arg600 of human ULK1 protein. Activation-state specific polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser757 of human ULK1 protein or residues surrounding Ser792 of human raptor 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.

Pathways

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