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69501
PhosphoPlus® Atg14 (Ser29) Antibody Duet
Primary Antibodies
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PhosphoPlus® Atg14 (Ser29) Antibody Duet #69501

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PhosphoPlus® Atg14 (Ser29) Antibody Duet: Image 1
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).
PhosphoPlus® Atg14 (Ser29) Antibody Duet: Image 2
Western blot analysis of extracts from HCT 116 and HCT 116/Atg14 shRNA knockout cells, untreated (-) or starved using Earle's Balanced Salt Solution (EBSS, 2 hr; +) and the ULK1 inhibitor SBI-0206965 #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.
PhosphoPlus® Atg14 (Ser29) Antibody Duet: Image 3
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).
PhosphoPlus® Atg14 (Ser29) Antibody Duet: Image 4
Western blot analysis of extracts from Saos-2 cells, untreated (-) or starved using Earle's Balanced 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).
PhosphoPlus® Atg14 (Ser29) Antibody Duet: Image 5
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.
PhosphoPlus® Atg14 (Ser29) Antibody Duet: Image 6
Flow cytometric analysis of HCT 116/Atg14 shRNA knockout cells treated with Torin 1 #14379 (250 nM, 18 hr; blue, lower-expressing) and HCT 116 wild-type cells treated with Torin 1 (green, higher-expressing) using Phospho-Atg14 (Ser29) (D4B8M) Rabbit mAb (solid lines) or a 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. HCT 116/Atg14 shRNA knockout cells were kindly provided by Dr. Do-Hyung Kim, University of Minnesota, Minneapolis, MN.
PhosphoPlus® Atg14 (Ser29) Antibody Duet: Image 7
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.
PhosphoPlus® Atg14 (Ser29) Antibody Duet: Image 8
Western blot analysis of extracts from various cell lines using Atg14 (D1A1N) Rabbit mAb.
PhosphoPlus® Atg14 (Ser29) Antibody Duet: Image 9
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.
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69501S		 1 Kit

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Product Description

PhosphoPlus® Duets from Cell Signaling Technology (CST) provide a means to assess protein activation status. Each Duet contains an activation-state and total protein antibody to your target of interest. These antibodies have been selected from CST's product offering based upon superior performance in specified applications.

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 is also 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 is directed by a number of autophagy-related (Atg) genes. These proteins are involved in the formation of autophagosomes, cytoplasmic vacuoles that are delivered to lysosomes for degradation. The class III type phosphoinositide 3-kinase (PI3K) Vps34 regulates vacuolar trafficking and autophagy (4,5). Multiple proteins associate with Vps34, including p105/Vps15, Beclin-1, UVRAG, Atg14, and Rubicon, to determine Vps34 function (6-12). Atg14 and Rubicon were identified based on their ability to bind to Beclin-1 and participate in unique complexes with opposing functions (9-12). Rubicon, which localizes to the endosome and lysosome, inhibits Vps34 lipid kinase activity; knockdown of Rubicon enhances autophagy and endocytic trafficking (11,12). In contrast, Atg14 localizes to autophagosomes, isolation membranes and ER, and can enhance Vps34 activity. Knockdown of Atg14 inhibits starvation-induced autophagy (11,12). The serine/threonine kinase ULK1 phosphorylates Atg14 at Ser29 to promote autophagosome formation (13).
  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. Corvera, S. (2001) Traffic 2, 859-66.
  5. Yan, Y. and Backer, J.M. (2007) Biochem Soc Trans 35, 239-41.
  6. Stack, J.H. et al. (1995) J Cell Biol 129, 321-34.
  7. Zeng, X. et al. (2006) J Cell Sci 119, 259-70.
  8. Liang, C. et al. (2006) Nat Cell Biol 8, 688-99.
  9. Itakura, E. et al. (2008) Mol Biol Cell 19, 5360-72.
  10. Sun, Q. et al. (2008) Proc Natl Acad Sci U S A 105, 19211-6.
  11. Zhong, Y. et al. (2009) Nat Cell Biol 11, 468-76.
  12. Matsunaga, K. et al. (2009) Nat Cell Biol 11, 385-96.
  13. Park, J.M. et al. (2016) Autophagy 12, 547-64.

Pathways & Proteins

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PhosphoPlus is a trademark of Cell Signaling Technology, Inc.