Revision 2

#46486Store at -20C

1 Kit

(6 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
ULK1 (D8H5) Rabbit mAb 8054 20 µl 150 kDa Rabbit IgG
Atg13 (D4P1K) Rabbit mAb 13273 20 µl 72 kDa Rabbit IgG
FIP200 (D10D11) Rabbit mAb 12436 20 µl 200 kDa Rabbit IgG
Atg101 (E1Z4W) Rabbit mAb 13492 20 µl 25 kDa Rabbit IgG
Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb 14202 20 µl 140-150 kDa Rabbit IgG
Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb 5869 20 µl 140-150 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

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.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibodies.

Background

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. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
  2. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ 12 Suppl 2, 1509-18.
  3. Levine, B. and Yuan, J. (2005) J Clin Invest 115, 2679-88.
  4. Ganley, I.G. et al. (2009) J Biol Chem 284, 12297-305.
  5. Hosokawa, N. et al. (2009) Mol Biol Cell 20, 1981-91.
  6. Jung, C.H. et al. (2009) Mol Biol Cell 20, 1992-2003.
  7. Kim, J. et al. (2011) Nat Cell Biol 13, 132-41.
  8. Mercer, C.A. et al. (2009) Autophagy 5, 649-62.
  9. Hosokawa, N. et al. (2009) Autophagy 5, 973-9.
  10. Egan, D.F. et al. (2011) Science 331, 456-61.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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    Revision 2
    #46486

    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit

    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 1 Expand Image
    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.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 2 Expand Image
    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.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from various cell lines using FIP200 (D10D11) Rabbit mAb.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from RD, PANC-1, and A20 cells using Atg13 (D4P1K) Rabbit mAb.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from various cell lines using Atg101 (E1Z4W) Rabbit mAb.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 6 Expand Image
    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).
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 7 Expand Image
    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.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 8 Expand Image
    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.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 9 Expand Image
    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.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 10 Expand Image
    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).
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 11 Expand Image
    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).
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 12 Expand Image
    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).
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from various cell lines using Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 14 Expand Image
    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.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of extracts from various cell lines using ULK1 (D8H5) Rabbit mAb.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 16 Expand Image
    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.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 17 Expand Image
    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.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 18 Expand Image
    Confocal immunofluorescent analysis of MCF7 cells, untreated (left), untreated and post-processed with λ-phosphatase (center) or treated with Torin 1 #14379 (250 nM, 5 hr; right), using Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb. Blue = Hoechst 33342 #4082 (fluorescent DNA dye).
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 19 Expand Image
    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.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 20 Expand Image
    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).
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 21 Expand Image
    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.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 22 Expand Image
    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.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 23 Expand Image
    Flow cytometric analysis of SCLC-21H cells, treated with Torin 1 #14379 (250 nM, 2 hr; blue) or untreated (green) using Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Autophagy Induction (ULK1 Complex) Antibody Sampler Kit: Image 24 Expand Image
    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).