Cell Signaling Technology

Product Pathways - Autophagy Signaling

Autophagy Induction (ULK1 Complex) Antibody Sampler Kit #46486

REACTIVITY

No. Size Price
46486T 1 Kit ( 6 x 20 µl ) ¥5,301.00 现货查询 购买询价
Kit Includes Quantity Applications Reactivity Homology† MW (kDa) Isotype
FIP200 (D10D11) Rabbit mAb #12436 20 µl W,IP, H,M, Mk, 200 Rabbit IgG
Atg13 (D4P1K) Rabbit mAb #13273 20 µl W,IP, H,M,R, 72 Rabbit IgG
Atg101 (E1Z4W) Rabbit mAb #13492 20 µl W, H,M,R,Mk, 25 Rabbit IgG
Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb #14202 20 µl W,IP, H,M,R, 140-150 Rabbit IgG
Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb #5869 20 µl W,IP, H,M, R, 140-150 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl W, Goat
ULK1 (D8H5) Rabbit mAb #8054 20 µl W,IP, H,M,R,Mk, 150 Rabbit IgG

Specificity / Sensitivity

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).

Source / Purification

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).

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.

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

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

Western Blotting

Western Blotting

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).

Western Blotting

Western Blotting

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

Western Blotting

Western Blotting

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).

Western Blotting

Western Blotting

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.

IP

IP

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.

Western Blotting

Western Blotting

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

Western Blotting

Western Blotting

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).

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

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).

Western Blotting

Western Blotting

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

Western Blotting

Western Blotting

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).

Western Blotting

Western Blotting

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

Western Blotting

Western Blotting

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).

Western Blotting

Western Blotting

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

Western Blotting

Western Blotting

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

Western Blotting

Western Blotting

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

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

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).

Western Blotting

Western Blotting

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

Western Blotting

Western Blotting

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

Western Blotting

Western Blotting

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).

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).

Application References

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