Revision 1

#59187Store at -20C

1 Kit

(6 x 20 microliters)

Cell Signaling Technology

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

Support: 877-678-TECH (8324)

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Beclin-1 (D40C5) Rabbit mAb 3495 20 µl 60 kDa Rabbit IgG
PI3 Kinase Class III (D9A5) Rabbit mAb 4263 20 µl 100 kDa Rabbit 
PIK3R4 Antibody 14580 20 µl 153 kDa Rabbit 
Atg14 (D1A1N) Rabbit mAb 96752 20 µl 65 kDa Rabbit IgG
UVRAG (D2Q1Z) Rabbit mAb 13115 20 µl 90 kDa Rabbit IgG
Rubicon (D9F7) Rabbit mAb 8465 20 µl 130 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 Beclin-1 Complex Antibody Sampler Kit provides an economical means of detecting proteins that are part of the Beclin-1 complexes. The kit includes enough antibodies to perform two western blot experiments with each 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 antibody.

Background

A number of studies have identified distinct complexes involving Beclin-1 and PI3K Kinase Class III with specific roles in autophagy and vesicle trafficking (1,2). These complexes commonly contain Beclin-1, PI3KC3/VSP34, and PIK3R4/VPS15 and function to catalyze the phosphorylation of phosphatidylinositol at the D3 position, producing phosphatidylinositol-3-phosphate. Specificity of PI3KC3 activity is regulated by additional binding partners. Complex 1 contains Atg14 which is required for early stages of autophagosome nucleation (3,4). Complex 2 lacks Atg14, but instead contains UVRAG, and is important for autophagosome maturation and endocytic trafficking (4-6). A third complex, containing both UVRAG and Rubicon, negatively regulates canonical autophagy (7,8). Importantly, this complex containing Rubicon is critical for a related process of LC3-associated phagocytosis (LAP) in which extracellular pathogens binding to cell surface receptors are engulfed by a single membrane phagosome and degraded by the lysosome (9,10).

  1. Levine, B. and Kroemer, G. (2019) Cell 176, 11-42.
  2. Mei, Y. et al. (2016) Protein Sci 25, 1767-85.
  3. Sun, Q. et al. (2008) Proc Natl Acad Sci U S A 105, 19211-6.
  4. Itakura, E. et al. (2008) Mol Biol Cell 19, 5360-72.
  5. Liang, C. et al. (2006) Nat Cell Biol 8, 688-99.
  6. Liang, C. et al. (2008) Nat Cell Biol 10, 776-87.
  7. Matsunaga, K. et al. (2009) Nat Cell Biol 11, 385-96.
  8. Zhong, Y. et al. (2009) Nat Cell Biol 11, 468-76.
  9. Martinez, J. et al. (2015) Nat Cell Biol 17, 893-906.
  10. Heckmann, B.L. and Green, D.R. (2019) J Cell Sci 132(5). pii: jcs222984. doi: 10.1242/jcs.222984.

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.

    Limited Uses

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    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #59187

    Beclin-1 Complex Antibody Sampler Kit

    Beclin-1 Complex Antibody Sampler Kit: Image 1 Expand Image
    Western blot analysis of extracts from various cell lines using UVRAG (D2Q1Z) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). HCT 116 cells have been reported to contain a monoallelic frameshift mutation resulting in significantly reduced levels of endogenous UVRAG expression (11).
    Beclin-1 Complex Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from various cell lines using PIK3R4 Antibody.
    Beclin-1 Complex Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from various cell lines using Beclin-1 (D40C5) Rabbit mAb.
    Beclin-1 Complex Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from mouse brain and C6 cells using PI3 Kinase Class III (D9A5) Rabbit mAb.
    Beclin-1 Complex Antibody Sampler Kit: Image 5 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.
    Beclin-1 Complex Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using Rubicon (D9F7) Rabbit mAb.
    Beclin-1 Complex Antibody Sampler Kit: Image 7 Expand Image
    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.
    Beclin-1 Complex Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing human UVRAG (hUVRAG; +), using UVRAG (D2Q1Z) Rabbit mAb.
    Beclin-1 Complex Antibody Sampler Kit: Image 9 Expand Image
    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.
    Beclin-1 Complex Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with human Rubicon (hRubicon, +), using Rubicon (D9F7) Rabbit mAb. Rubicon construct was kindly provided by Dr. Qing Zhong, University of California, Berkeley, CA.
    Beclin-1 Complex Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from various cell lines using Atg14 (D1A1N) Rabbit mAb.
    Beclin-1 Complex Antibody Sampler Kit: Image 12 Expand Image
    Immunoprecipitation of UVRAG from PANC-1 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or UVRAG (D2Q1Z) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using UVRAG (D2Q1Z) Rabbit mAb.
    Beclin-1 Complex Antibody Sampler Kit: Image 13 Expand Image
    Immunoprecipitation of Atg14 from Neuro2A 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.