Western blot analysis of extracts from various cell lines using Beclin-1 (D40C5) Rabbit mAb.
Western blot analysis of extracts from mouse brain and C6 cells using PI3 Kinase Class III (D9A5) Rabbit mAb.
Western blot analysis of extracts from various cell lines using PIK3R4 Antibody.
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 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).
Western blot analysis of extracts from various cell lines using Rubicon (D9F7) Rabbit mAb.
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 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 blot analysis of extracts from various cell lines using Atg14 (D1A1N) Rabbit mAb.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing human UVRAG (hUVRAG; +), using UVRAG (D2Q1Z) Rabbit mAb.
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.
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.
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 (D40C5) Rabbit mAb 3495||20 µl||
||H M R Mk||60||Rabbit IgG|
|PI3 Kinase Class III (D9A5) Rabbit mAb 4263||20 µl||
||H M R Mk||100||Rabbit|
|PIK3R4 Antibody 14580||20 µl||
||H M R||153||Rabbit|
|Atg14 (D1A1N) Rabbit mAb 96752||20 µl||
||H M R||65||Rabbit IgG|
|UVRAG (D2Q1Z) Rabbit mAb 13115||20 µl||
||H M||90||Rabbit IgG|
|Rubicon (D9F7) Rabbit mAb 8465||20 µl||
||H M||130||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
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.
Each antibody in the Beclin-1 Complex Antibody Sampler Kit detects endogenous levels of its target protein. Rubicon (D9F7) Rabbit mAb detects a band of unknown origin at 55 kDa.
Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Thr72 of human Beclin-1, Lys630 of human PI3 Kinase Class III, Arg70 of human Atg14, Gly502 of human UVRAG, and Leu210 of human Rubicon. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly825 of human PIK3R4 protein. Antibodies are purified by protein A and peptide affinity chromatography.
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).
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