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4445
Autophagy Antibody Sampler Kit
Primary Antibodies

Autophagy Antibody Sampler Kit #4445

Western Blotting Image 1

Western blot analysis of extracts from HeLa, NIH/3T3, and KNRK cells, untreated (-) or chloroquine-treated (50 μM, overnight; +), using LC3A/B (D3U4C) XP® Rabbit mAb #12741.

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Western Blotting Image 2

Western blot analysis of extracts from various cell lines using Atg5 (D5F5U) Rabbit mAb #12994.

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Western Blotting Image 3

Western blot analysis of extracts from various cell lines using Atg16L1 (D6D5) Rabbit mAb #8089.

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Western Blotting Image 4

Western blot analysis of extracts from various cell lines using Beclin-1 (D40C5) Rabbit mAb.

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Western Blotting Image 5

Western blot analysis of extracts from RD cells, untreated (-) or Torin 1-treated (250 nM, 4 hr; +), using LC3A/B (D3U4C) XP® Rabbit mAb.

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Western Blotting Image 6

Western blot analysis of extracts from various cell lines using Atg5 (D5F5U) Rabbit mAb.

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Western Blotting Image 7

Western blot analysis of extracts from various cell lines using Atg12 (D88H11) Rabbit mAb.

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Western Blotting Image 8

Western blot analysis of extracts from various cell lines using Atg16L1 (D6D5) Rabbit mAb.

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Western Blotting Image 9

Western blot analysis of extracts from various cell lines using Atg7 (D12B11) Rabbit mAb.

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Western Blotting Image 10

Western blot analysis of extracts from various cell lines using Atg3 Antibody.

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Western Blotting Image 11

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.

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Western Blotting Image 12

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.

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Western Blotting Image 13

Western blot analysis of extracts from HeLa, NIH/3T3, and KNRK cells, untreated (-) or chloroquine-treated (50 μM, overnight; +), using LC3A/B (D3U4C) XP® Rabbit mAb.

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Western Blotting Image 14

Western blot analysis of extracts from wild-type MEFs (wt) or MEFs from Atg5 knockouts (Atg5-/-) using Atg5 (D5F5U) Rabbit mAb (upper), or β-Actin (D6A8) Rabbit mAb #8457 (lower). Atg5-/- MEFs were kindly provided by Dr. Ramnik Xavier, Massachusetts General Hospital, Harvard Medical School, Boston, MA.

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Western Blotting Image 15

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with either a myc-tagged human Atg16L1β construct (hAtg16L1β-Myc; +) or a mouse Atg16L1α construct (mAtg16L1α; +), using Atg16L1 (D6D5) Rabbit mAb. The myc-tagged human Atg16L1β construct was kindly provided by Dr. Qing Zhong, University of California Berkeley.

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Western Blotting Image 16

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Atg7 siRNA I #6604 (+), using Atg7 (D12B11) Rabbit mAb (upper) or α-Tubulin (11Η10) Rabbit mAb #2125 (lower). The Atg7 (D12B11) Rabbit mAb confirms silencing of Atg7 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

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Western Blotting Image 17

Western blot analysis of extracts from HeLa cells, mock transfected or transfected with mouse Atg3, using Atg3 Antibody.

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IHC-P (paraffin) Image 18

Immunohistochemical analysis of paraffin-embedded mouse prostate using LC3A/B (D3U4C) XP® Rabbit mAb.

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IP Image 19

Immunoprecipitation of Atg5 from PANC-1 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Atg5 (D5F5U) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using Atg5 (D5F5U) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody to avoid cross-reactivity with rabbit IgG.

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IF-IC Image 20

Confocal immunofluorescent analysis of EBSS-starved PANC-1 cells using Atg16L1 (D6D5) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 21

Immunohistochemical analysis of paraffin-embedded NIH/3T3 cell pellets, control (left) or chloroquine-treated (right), using LC3A/B (D3U4C) XP® Rabbit mAb.

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IHC-P (paraffin) Image 22

Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using LC3A/B (D3U4C) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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Flow Cytometry Image 23

Flow cytometric analysis of HeLa cells, untreated (blue) or treated with chloroquine (50 µM, 16 hr) (green), using LC3A/B (D3U4C) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

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IF-IC Image 24

Confocal immunofluorescent analysis of HeLa (upper) and C2C12 (lower) cells, chloroquine-treated (50 μM, overnight; left), nutrient-starved with EBSS (3 hr, middle) or untreated (right) using LC3A/B (D3U4C) XP® Rabbit mAb (green) and β-Actin (13E5) Rabbit mAb (Alexa Fluor® 555 Conjugate) #8046 (red). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Beclin-1 (D40C5) Rabbit mAb 3495 20 µl
  • WB
  • IP
H M R Mk 60 Rabbit IgG
LC3A/B (D3U4C) XP® Rabbit mAb 12741 20 µl
  • WB
  • IHC
  • IF
  • F
H M R 14, 16 Rabbit IgG
Atg5 (D5F5U) Rabbit mAb 12994 20 µl
  • WB
  • IP
H M R 55 Rabbit IgG
Atg12 (D88H11) Rabbit mAb 4180 20 µl
  • WB
  • IP
H M R Mk 16, 55 Rabbit IgG
Atg16L1 (D6D5) Rabbit mAb 8089 20 µl
  • WB
  • IP
  • IF
H M R 66, 68 Rabbit IgG
Atg7 (D12B11) Rabbit mAb 8558 20 µl
  • WB
  • IP
H M R 78 Rabbit IgG
Atg3 Antibody 3415 20 µl
  • WB
H M R Mk 40 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Autophagy Antibody Sampler Kit provides an economical means to investigate the molecular machinery of autophagy within the cell. The kit contains enough primary and secondary antibodies to perform two Western blot experiments.

Each antibody in the Autophagy Antibody Sampler Kit detects its respective target at endogenous levels. Both the Atg5 and Atg12 rabbit monoclonal antibodies recognize the Atg12-Atg5 conjugate.

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu44 of human LC3B, Thr72 of human Beclin-1, Leu265 of human Atg5, Ser36 of human Atg12, or near the amino termini of human Atg7 and Atg16L1. Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues near the amino-termini of human Atg3. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

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. Formation of the autophagosome involves a ubiquitin-like conjugation system in which Atg12 is covalently bound to Atg5 and targeted to autophagosome vesicles (4-6). This conjugation reaction is mediated by the ubiquitin E1-like enzyme Atg7 and the E2-like enzyme Atg10 (7,8).

  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. Mizushima, N. et al. (1998) J Biol Chem 273, 33889-92.
  5. Mizushima, N. et al. (1998) Nature 395, 395-8.
  6. Suzuki, K. et al. (2001) EMBO J 20, 5971-81.
  7. Tanida, I. et al. (1999) Mol Biol Cell 10, 1367-79.
  8. Shintani, T. et al. (1999) EMBO J 18, 5234-41.
Entrez-Gene Id
9140 , 55054 , 64422 , 9474 , 10533 , 8678 , 84557 , 81631
Swiss-Prot Acc.
O94817 , Q676U5 , Q9NT62 , Q9H1Y0 , O95352 , Q14457 , Q9H492 , Q9GZQ8
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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