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35277
AMPK Substrate Antibody Sampler Kit
Primary Antibodies

AMPK Substrate Antibody Sampler Kit #35277

Western Blotting Image 1

Western blot analysis of extracts from serum-starved NCI-H2228 cells, untreated (-) or treated with phenformin (5 mM, 1 hr; +), using Phospho-AMPKα (Thr172) (D4D6D) Rabbit mAb (upper) or AMPKα (D63G4) Rabbit mAb #5832 (lower).

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Western Blotting Image 2

Western blot analysis of extracts from HeLa, K-562, C6, and Neuro-2a cells using AMPKα (D5A2) Rabbit mAb.

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Western Blotting Image 3

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.

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Western Blotting Image 4

Western blot analysis of extracts from various cell lines using ULK1 (D8H5) Rabbit mAb.

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Western Blotting Image 5

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.

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Western Blotting Image 6

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

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Western Blotting Image 7

Western blot analysis of extracts from various cell lines using Beclin-1 (D40C5) Rabbit mAb.

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Western Blotting Image 8

Western blot analysis of extracts from MCF7 and KARPAS-299 cells, untreated (-) or treated with carbonyl cyanide 3-chlorophenylhdrazone (CCCP, 100 μM, 2 hr; +), using Phospho-Beclin-1 (Ser93) (D9A5G) Rabbit mAb (upper) and Beclin-1 (D40C5) Rabbit mAb #3495 (lower). Cell line source: Dr Abraham Karpas at the University of Cambridge.

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Western Blotting 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.

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IHC-P (paraffin) Image 10

Immunohistochemical analysis of paraffin-embedded NCI-H2228 cell pellets, untreated (left) or phenformin-treated (right), using Phospho-AMPKα (Thr172) (D4D6D) Rabbit mAb.

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Western Blotting Image 11

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.

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Western Blotting Image 12

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).

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Western Blotting Image 13

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

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Western Blotting Image 14

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® Beclin-1 siRNA I #6222 (+) or SignalSilence® Beclin-1 siRNA II (+), using Beclin-1 (D40C5) XP® Rabbit mAb #3495 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Beclin-1 (D40C5) XP® Rabbit mAb confirms silencing of Beclin-1 expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of Beclin-1 siRNA.

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Western Blotting Image 15

Western blot analysis of extracts from 293T cells, untreated (-) or treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 100 μM, 2 hr; +) and mock transfected (-) or transfected with a construct expressing full-length human Beclin-1 (hBeclin-1; +), using Phospho-Beclin-1 (Ser93) (D9A5G) Rabbit mAb (upper) and Beclin-1 (D40C5) Rabbit mAb #3495 (lower).

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IHC-P (paraffin) Image 16

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-AMPKα (Thr172) (D4D6D) Rabbit mAb, control (left) or λ phosphatase-treated (right).

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Western Blotting Image 17

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).

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IP Image 18

Immunoprecipitation of phospho-Beclin-1 (Ser93) from carbonyl cyanide 3-chlorophenyldydrazone (CCCP)-treated KARPAS-299 cells (100 μM, 2 hr) using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-Beclin-1 (Ser93) (D9A5G) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using Phospho-Beclin-1 (Ser93) (D9A5G) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody to avoid cross reactivity with IgG heavy chain. Cell line source: Dr Abraham Karpas at the University of Cambridge.

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IHC-P (paraffin) Image 19

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Phospho-AMPKα (Thr172) (D4D6D) Rabbit mAb.

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IHC-P (paraffin) Image 20

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-AMPKα (Thr172) (D4D6D) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-AMPKα (Thr172) (D4D6D) Rabbit mAb 50081 20 µl
  • WB
  • IP
  • IHC
H M R 62 Rabbit IgG
AMPKα (D5A2) Rabbit mAb 5831 20 µl
  • WB
  • IP
H M R Mk B 62 Rabbit 
Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb 5869 20 µl
  • WB
  • IP
H M 140-150 Rabbit IgG
ULK1 (D8H5) Rabbit mAb 8054 20 µl
  • WB
  • IP
H M R Mk 150 Rabbit IgG
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 
Beclin-1 (D40C5) Rabbit mAb 3495 20 µl
  • WB
  • IP
H M R Mk 60 Rabbit IgG
Phospho-Beclin-1 (Ser93) (D9A5G) Rabbit mAb 14717 20 µl
  • WB
  • IP
H 60 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The AMPK Substrate Antibody Sampler Kit provides an economical means of detecting total and phosphorylated substrates of AMPK. The kit provides enough antibody to perform two western blots with each primary antibody.

Each antibody in the AMPK Substrate Antibody Sampler Kit detects endogenous levels of its target protein. Phospho-AMPKα (Thr172) (D4D6D) Rabbit mAb and

AMPKα (D5A2) Rabbit mAb detects both α1 and α2 isoforms of the catalytic subunit. Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb detects endogenous levels of ULK1 only when phosphorylated at Ser555 of mouse ULK1 (equivalent to Ser556 of human ULK1). Bands of unknown origin are detected between 90 and 100 kDa. Phospho-Raptor (Ser792) Antibody detects non-specific signals of various molecular weights. Phospho-Beclin-1 (Ser93) (D9A5G) Rabbit mAb recognizes single phosphorylation of Ser93 to a greater extent than dual phosphorylation at Ser93 and Ser96. This antibody may also weakly detect an unidentified band at approximately 70 kDa.

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide and phosphopeptides corresponding to residues surrounding Ser172 of human AMPKα protein, Arg21 of human AMPKα protein, Ser555 of mouse ULK1 protein (equivalent to Ser556 of human ULK1), Arg600 of human ULK1, Glu11 of human Raptor, Ser93 of human Beclin-1 (Ser91 in mouse), and Thr72 of human Beclin-1. Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to the sequence surrounding Ser792 of human raptor. Antibodies are purified by peptide affinity chromatography. .

AMP-activated protein kinase (AMPK) is highly conserved from yeast to plants and animals and plays a key role in the regulation of energy homeostasis (1). AMPK is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits, each of which is encoded by two or three distinct genes (α1, 2; β1, 2; γ1, 2, 3) (2). The kinase is activated by an elevated AMP/ATP ratio due to cellular and environmental stress, such as heat shock, hypoxia, and ischemia (1). The tumor suppressor LKB1, in association with accessory proteins STRAD and MO25, phosphorylates AMPKα at Thr172 in the activation loop, and this phosphorylation is required for AMPK activation (3-5).

AMPK phosphorylates a number of targets controlling cellular processes such as metabolism, cell growth, and autophagy (6). It suppresses the activity of the mammalian target of rapamycin (mTOR), that plays a key role in promoting cell growth. The regulatory associated protein of mTOR (Raptor) was identified as an mTOR binding partner that mediates mTOR signaling to downstream targets (7,8). Raptor binds to mTOR substrates, including 4E-BP1 and p70 S6 kinase, through their TOR signaling (TOS) motifs and is required for mTOR-mediated phosphorylation of these substrates (9,10). AMPK directly phosphorylates Raptor at Ser722/Ser792, and this phosphorylation is essential for inhibition of the raptor-containing mTOR complex 1 (mTORC1) and induces cell cycle arrest when cells are stressed for energy (11). AMPK also promotes autophagy by directly phosphorylating ULK1 (11,12). ULK1 is a Ser/Thr kinase required for the Initiation and formation of the autophagosome. AMPK, activated during low nutrient conditions, directly phosphorylates ULK1 at multiple sites including Ser317, Ser555, and Ser777 (11,12). 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 (11). AMPK can also directly phosphorylate Beclin-1, a component of the complex downstream of ULK1 in autophagosome formation that activates the class III phosphatidylinositol 3-kinase VPS34. AMPK phosphorylates Beclin-1 at Ser93 and Ser96 residues in human, which correspond to murine Ser91 and Ser94 (14).

  1. Hardie, D.G. (2004) J Cell Sci 117, 5479-87.
  2. Carling, D. (2004) Trends Biochem Sci 29, 18-24.
  3. Hawley, S.A. et al. (1996) J Biol Chem 271, 27879-87.
  4. Lizcano, J.M. et al. (2004) EMBO J 23, 833-43.
  5. Shaw, R.J. et al. (2004) Proc Natl Acad Sci USA 101, 3329-35.
  6. Mihaylova, M.M. and Shaw, R.J. (2011) Nat Cell Biol 13, 1016-23.
  7. Hara, K. et al. (2002) Cell 110, 177-189.
  8. Kim, D.H. et al. (2002) Cell 110, 163-175.
  9. Beugnet, A. et al. (2003) J. Biol. Chem. 278, 40717-40722.
  10. Nojima, H. et al. (2003) J. Biol. Chem. 278, 15461-15464.
  11. Gwinn, D.M. et al. (2008) Mol Cell 30, 214-26.
  12. Kim, J. et al. (2011) Nat Cell Biol 13, 132-41.
  13. Egan, D.F. et al. (2011) Science 331, 456-61.
  14. Kim, J. et al. (2013) Cell 152, 290-303.
Entrez-Gene Id
5562 , 5563 , 8678 , 57521 , 8408
Swiss-Prot Acc.
Q13131 , P54646 , Q14457 , Q8N122 , O75385
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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