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46486
Autophagy Induction (ULK1 Complex) Antibody Sampler Kit

Autophagy Induction (ULK1 Complex) Antibody Sampler Kit #46486

Western Blotting Image 1

Western blot analysis of extracts from various cell lines using FIP200 (D10D11) Rabbit mAb #12436.

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Western Blotting Image 2

Western blot analysis of extracts from RD, PANC-1, and A20 cells using Atg13 (D4P1K) Rabbit mAb #13273.

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Western Blotting Image 3

Western blot analysis of extracts from various cell lines using Atg101 (E1Z4W) Rabbit mAb #13492.

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Western Blotting Image 4

Western blot analysis of extract from A172 cells, untreated (-) or treated with mTOR inhibitors, either Torin-1 (250 nM, 5 hrs), Torin-2 (250 nM, 5 hrs), or INK128 (250 nM, 5 hours) using Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb #14202 (upper) or ULK1 (D8H5) Rabbit mAb #8054 (lower).

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Western Blotting Image 5

Western blot analysis of extracts from various cell lines using Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb #14202.

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Western Blotting Image 6

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 #5869.

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Western Blotting Image 7

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 #5869 (upper) or ULK1 (D8H5) Rabbit mAb #8054 (lower).

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Western Blotting Image 8

Western blot analysis of extracts from various cell lines using ULK1 (D8H5) Rabbit mAb #8054.

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Western Blotting Image 9

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

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Western Blotting Image 10

Western blot analysis of extracts from RD, PANC-1, and A20 cells using Atg13 (D4P1K) Rabbit mAb.

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Western Blotting Image 11

Western blot analysis of extracts from various cell lines using FIP200 (D10D11) Rabbit mAb.

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Western Blotting Image 12

Western blot analysis of extracts from various cell lines using Atg101 (E1Z4W) Rabbit mAb.

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Western Blotting Image 13

Western blot analysis of extract from A172 cells, untreated (-) or treated with mTOR inhibitors, either Torin-1 (250 nM, 5 hrs), Torin-2 (250 nM, 5 hrs), or INK128 (250 nM, 5 hours) using Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb (upper) or ULK1 (D8H5) Rabbit mAb #8054 (lower).

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Western Blotting Image 14

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 15

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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Western Blotting Image 16

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 17

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Atg13 (hAtg13-Myc/DDK; +), using Atg13 (D4P1K) Rabbit mAb (upper) or Myc-Tag (71D10) Rabbit mAb #2278 (lower).

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Western Blotting Image 18

Western blot analysis of extracts from A172 cells, untreated (-) or chloroquine-treated (50 μM, overnight; +) using FIP200 (D10D11) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).

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Western Blotting Image 19

Western blot analysis of extracts from 293 cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Atg101 protein (hAtg101-Myc/DDK; +), using Atg101 (E1Z4W) Rabbit mAb (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).

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Western Blotting Image 20

Western blot analysis of extracts from various cell lines using Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb.

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Western Blotting Image 21

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 22

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Atg13 siRNA I #12043 (+), using Atg13 (D4P1K) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The Atg13 (D4P1K) Rabbit mAb confirms silencing of Atg13 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

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Western Blotting Image 23

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expresssing full-length human FIP200 (hFIP200; +), using FIP200 (D10D11) Rabbit mAb.

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Western Blotting Image 24

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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Western Blotting Image 25

Immunoprecipitation of Atg13 from RD cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype control #3900 (lane 2) or Atg13 (D4P1K) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Atg13 (D4P1K) Rabbit mAb.

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IP Image 26

Immunoprecipitation of FIP200 from JJN-3 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or FIP200 (D10D11) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using FIP200 (D10D11) Rabbit mAb.

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
ULK1 (D8H5) Rabbit mAb 8054 20 µl
  • WB
  • IP
H M R Mk 150 Rabbit IgG
Atg13 (D4P1K) Rabbit mAb 13273 20 µl
  • WB
  • IP
H M R 72 Rabbit IgG
FIP200 (D10D11) Rabbit mAb 12436 20 µl
  • WB
  • IP
H M 200 Rabbit IgG
Atg101 (E1Z4W) Rabbit mAb 13492 20 µl
  • WB
H M R Mk 25 Rabbit IgG
Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb 14202 20 µl
  • WB
  • IP
H M R 140-150 Rabbit IgG
Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb 5869 20 µl
  • WB
  • IP
H M 140-150 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Autophagy Induction (ULK1 Complex) Antibody Sampler Kit provides an economical means of detecting target proteins in the ULK1 complex. The kit contains enough antibody to perform at least two western blot experiments per primary antibody.

ULK1 (D8H5) Rabbit mAb, Atg13 (D4P1K) Rabbit mAb, FIP200 (D10D11) Rabbit mAb, and Atg101 (E1Z4W) Rabbit mAb recognize total endogenous levels of the corresponding target proteins irrespective of phosphorylation state. 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-ULK1 (Ser757) (D7O6U) Rabbit mAb recognizes endogenous levels of ULK1 protein only when phosphorylated at Ser757 of mouse ULK1 (equivalent to Ser758 of human ULK1).

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg600 of human ULK1 protein, residues surrounding Asp462 of human Atg13 protein, residues surrounding Val177 of human Atg101 protein, or residues near the carboxy terminus of human FIP200 protein. Phospho-specific monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser555 of mouse ULK1 protein (equivalent to Ser556 of human ULK1) or residues surrounding Ser757 of mouse ULK1 protein (equivalent to Ser758 of human ULK1).

Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation but has also been associated with a number of physiological processes including development, differentiation, neurodegeneration, infection, and cancer (3). The molecular machinery of autophagy was largely discovered in yeast and referred to as autophagy-related (Atg) genes. ULK1, Atg13, and FIP200 form a complex that localizes to autophagic isolation membranes and regulates autophagosome biogenesis (4-6). mTOR phosphorylates both Atg13 and ULK1, suppressing ULK1 kinase activity and autophagy (5-7). Interaction between Atg101 and Atg13 can be important for the stability and basal phosphorylation of Atg13 and ULK1 (8,9). AMPK, activated during low nutrient conditions, directly phosphorylates ULK1 at multiple sites including Ser317, Ser555, and Ser777 (7,10). Conversely, mTOR, which is a regulator of cell growth and is an inhibitor of autophagy, phosphorylates ULK1 at Ser757 and disrupts the interaction between ULK1 and AMPK (7).

  1. Jung, C.H. et al. (2009) Mol Biol Cell 20, 1992-2003.
  2. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot. Cell 1, 11-21.
  3. Mercer, C.A. et al. (2009) Autophagy 5, 649-62.
  4. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ. 12 Suppl 2, 1509-18.
  5. Hosokawa, N. et al. (2009) Autophagy 5, 973-9.
  6. Levine, B. and Yuan, J. (2005) J. Clin. Invest. 115, 2679-88.
  7. Ganley, I.G. et al. (2009) J Biol Chem 284, 12297-305.
  8. Hosokawa, N. et al. (2009) Mol Biol Cell 20, 1981-91.
  9. Kim, J. et al. (2011) Nat Cell Biol 13, 132-41.
  10. Egan, D.F. et al. (2011) Science 331, 456-61.
Entrez-Gene Id
60673 , 9776 , 9821 , 8408
Swiss-Prot Acc.
Q9BSB4 , O75143 , Q8TDY2 , O75385
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Tween is a registered trademark of ICI Americas, Inc.

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Product # Size Price
46486T
1 Kit (6 x 20 µl) $ 423.0