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#85493 ULK1 Substrate Antibody Sampler Kit

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2019年3月20日11時40分 現在
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#85493T1 Kit115,000
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キット内容 容量 用途 種交差性 ホモロジー† 検出分子量 アイソタイプ
Phospho-Atg14 (Ser29) (D4B8M) Rabbit mAb #92340 20 µl WB, IF-IC H, M, R 65 Rabbit IgG
Atg14 (D1A1N) Rabbit mAb #96752 20 µl WB, IP H, M, R 65 Rabbit IgG
Phospho-Beclin-1 (Ser15) (D4B7R) Rabbit mAb #84966 20 µl WB H, M 60 Rabbit IgG
Beclin-1 (D40C5) Rabbit mAb #3495 20 µl WB, IP H, M, R, Mk 60 Rabbit IgG
Phospho-Atg13 (Ser355) (D6J1W) Rabbit mAb #26839 20 µl WB, IP H M, R 72 Rabbit IgG
Atg13 (D4P1K) Rabbit mAb #13273 20 µl WB, IP H, M, R 72 Rabbit IgG
ULK1 (D8H5) Rabbit mAb #8054 20 µl WB, IP H, M, R, Mk 150 Rabbit IgG
Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb #14202 20 µl WB, IP, IF-IC H, M, R 140-150 Rabbit IgG
Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb #5869 20 µl WB, IP H, M R 140-150 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl WB Goat

†Species predicted to react based on 100% sequence homology.

Applications Key: W=Western Blotting, IF-IC=Immunofluorescence (Immunocytochemistry), IP=Immunoprecipitation
Reactivity Key: H=Human, M=Mouse, R=Rat, Mk=Monkey

特異性・感度
Phospho-Beclin-1 (Ser15) (D4B7R) Rabbit mAb and Phospho-Atg13 (Ser555) (E6H1W) Rabbit mAb are only recommended for transfected levels of their specific targets. Phospho-Atg13 (Ser555) (E6H1W) Rabbit mAb can weakly detect endogenous levels of phosphorylated Atg13, but also reacts with a band of unknown origin at 25 kDa. All other antibodies in the kit can detect endogenous levels of their specific targets. Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb detects a band of unknown origin between 90 and 100 kDa.
使用抗原
Monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to residues surrounding Arg70 of human Atg14, Thr72 of human Beclin-1, Asp462 of human Atg13, and Arg600 of human ULK1. Phosphorylation-specific monoclonal antibodies are produced by immunizing rabbits with synthetic phospho-peptides corresponding to Ser29 of Atg14, Ser15 of human Beclin-1, Ser355 of human Atg13 (Ser318 of isoform 2 of Atg13), Ser555 of mouse ULK1 (equivalent to Ser556 of human ULK1), and Ser757 of mouse ULK1 (equivalent to Ser758 of human ULK1).

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貯法
-20℃
社内データ

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 various cell lines using Beclin-1 (D40C5) 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), 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 various cell lines using ULK1 (D8H5) 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 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 293T cells, mock transfected (-) or transfected with constructs expressing full-length human Beclin-1 protein (hBeclin-1; +), mouse Beclin-1 protein (mBeclin-1; +), or mouse ULK1 protein (mULK1; +), using Phospho-Beclin-1 (Ser15) (D4B7R) Rabbit mAb (upper), Beclin-1 (D40C5) Rabbit mAb #3495 (middle) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from HCT 116 and HCT 116/Atg14 shRNA knockout cells using Atg14 (D1A1N) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #84576 (lower). HCT 116/Atg14 shRNA cells were kindly provided by Dr. Do-Hyung Kim, University of Minnesota, Minneapolis, MN.

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; +) or mouse ULK1 (mULK1; +), using Phospho-Atg13 (Ser355) (D6J1W) Rabbit mAb.


Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing GFP-tagged human Atg14 protein (hAtg14-GFP; +) or mouse ULK1 protein (mULK1; +), using Phospho-Atg14 (Ser29) (D4B8M) Rabbit mAb (upper), Atg14 (D1A1N) Rabbit mAb (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Western Blotting

Western Blotting

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.

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 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 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 various cell lines using Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Atg14 (D1A1N) Rabbit mAb.

IP

IP

Immunoprecipitation of Phospho-Atg13 (Ser355) from extracts of PANC-1 cells starved with Hank's Balanced Salt Solution (HBSS) (2 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-Atg13 (Ser355) (D6J1W) Rabbit mAb. Western blot was performed using Phospho-Atg13 (Ser355) (D6J1W) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from HCT 116 and HCT 116/Atg14 shRNA knockout cells, untreated (-) or starved using Earles Basic Salt Solution (EBSS, 2 hr; +) and the ULK1 inhibitor SBI-020695 #29089 (50 μM, 2 hr; +) as indicated, using Phospho-Atg14 (Ser29) (D4B8M) Rabbit mAb (upper), Atg14 (D1A1N) Rabbit mAb #96752 (middle), or β-Actin (D6A8) Rabbit mAb (lower). HCT 116/Atg14 shRNA knockout cells were kindly provided by Dr. Do-Hyung Kim, University of Minnesota, Minneapolis, MN.


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

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).

IP

IP

Immunoprecipitation of Atg14 from HCT 116 cell extracts. Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Atg14 (D1A1N) Rabbit mAb. Western blot was performed using Atg14 (D1A1N) Rabbit mAb. A confirmation specific secondary antibody was used to avoid reactivity with IgG.


Western Blotting

Western Blotting

Western blot analysis of extracts from HCT 116 cells, untreated (-) or treated with lambda-phosphatase and calf intestinal phosphatase (λ-phosphatase/CIP; +), using Phospho-Atg14 (Ser29) (D4B8M) Rabbit mAb (upper), Atg14 (D1A1N) Rabbit mAb #96752 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).

IF-IC

IF-IC

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).

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 Saos-2 cells, untreated (-) or starved using Earles Basic Salt Solution (EBSS, 2 hr), using Phospho-Atg14 (Ser29) (D4B8M) Rabbit mAb (upper), Atg14 (D1A1N) Rabbit mAb #96752 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HCT 116 Atg14 wild-type cells, untreated (left, low-expressing) or treated with Torin 1 #14379 (250 nM, 2 hr; middle-left, high-expressing), HCT 116/Atg14 shRNA knockout cells treated with Torin 1 (middle-right, negative), or HCT 116 Atg14 wild-type cells post-processed with λ-phosphatase (2 hr; right, negative), using Phospho-Atg14 (Ser29) (D4B8M) Rabbit mAb (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue). HCT 116/Atg14 shRNA knockout cells were kindly provided by Dr. Do-Hyung Kim, University of Minnesota, Minneapolis, MN.

バックグラウンド

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).

AMPK, activated during low nutrient conditions, directly phosphorylates ULK1 at multiple sites, including Ser317, Ser555, and Ser777 (17,18). 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 (17). ULK1 has been shown to phoshorylate several targets in the autophagy pathway, including Ser29 of Atg14, Ser15 of Beclin-1, and Ser355 of Atg13 (19-22).

使用例
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関連製品
12630   SignalFire™ Plus ECL Reagent
12757   SignalFire™ Elite ECL Reagent
13273   Atg13 (D4P1K) Rabbit mAb
14202   Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb
26839   Phospho-Atg13 (Ser355) (D6J1W) Rabbit mAb
3495   Beclin-1 (D40C5) Rabbit mAb
5869   Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb
6883   SignalFire™ ECL Reagent
7074   Anti-rabbit IgG, HRP-linked Antibody
7727   Biotinylated Protein Ladder Detection Pack
8054   ULK1 (D8H5) Rabbit mAb
84966   Phospho-Beclin-1 (Ser15) (D4B7R) Rabbit mAb
92340   Phospho-Atg14 (Ser29) (D4B8M) Rabbit mAb
96752   Atg14 (D1A1N) Rabbit mAb

Tween is a registered trademark of ICI Americas, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
ProLong is a registered trademark of Life Technologies Corporation.
SignalSilence is a registered trademark of Cell Signaling Technology, Inc.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

本製品は試験研究用です。

ULK1 Substrate Antibody Sampler Kit

Metabolic Reprogramming in Disease

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