Revision 2

#46486

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Mol. Wt Isotype/Source
ULK1 (D8H5) Rabbit mAb805420 µl150 kDaRabbit IgG
Atg13 (D4P1K) Rabbit mAb1327320 µl72 kDaRabbit IgG
FIP200 (D10D11) Rabbit mAb1243620 µl200 kDaRabbit IgG
Atg101 (E1Z4W) Rabbit mAb1349220 µl25 kDaRabbit IgG
Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb1420220 µl140-150 kDaRabbit IgG
Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb586920 µl140-150 kDaRabbit IgG
Anti-rabbit IgG, HRP-linked Antibody7074100 µlGoat

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).

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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专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品 , (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Western blot analysis of extracts from various cell lines using FIP200 (D10D11) Rabbit mAb.
undefined Image 1: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
Western blot analysis of extracts from RD, PANC-1, and A20 cells using Atg13 (D4P1K) Rabbit mAb.
undefined Image 2: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using Atg101 (E1Z4W) Rabbit mAb.
undefined Image 3: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
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).
undefined Image 4: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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.
undefined Image 5: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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.
undefined Image 6: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
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.
undefined Image 7: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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).
undefined Image 8: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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).
undefined Image 9: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
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).
undefined Image 10: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using Phospho-ULK1 (Ser757) (D7O6U) Rabbit mAb.
undefined Image 11: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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.
undefined Image 12: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Western blot analysis of extracts from various cell lines using ULK1 (D8H5) Rabbit mAb.
undefined Image 13: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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.
undefined Image 14: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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.
undefined Image 15: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
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).
undefined Image 16: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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.
undefined Image 17: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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).
undefined Image 18: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
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.
undefined Image 19: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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.
undefined Image 20: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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.
undefined Image 21: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
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).
undefined Image 22: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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.
undefined Image 23: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
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.
undefined Image 24: Autophagy Induction (ULK1 Complex) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.