Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Apoptosis / Autophagy

LC3A (D50G8) XP® Rabbit mAb #4599

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IHC-P IF-IC F H M R (Mk) (Dg) Endogenous 14, 16 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Dg=Dog
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

* Product-specific protocol.

Specificity / Sensitivity

LC3A (D50G8) XP® Rabbit mAb detects endogenous levels of total LC3A protein. This antibody may also react with LC3B.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human LC3A.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated or chloroquine-treated (50 μM, overnight), using LC3A (D50G8) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human glioblastoma multiforme using LC3A (D50G8) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using LC3A (D50G8) XP® Rabbit mAb (left) orRabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, control (left) or choloroquine-treated (right), using LC3A (D50G8) XP® Rabbit mAb.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Chloroquine-treated HeLa cells, using LC3A (D50G8) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or chloroquine-treated (right), using LC3A (D50G8) XP® Rabbit mAb (green). Actin filaments were labeled using DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).


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, neurodegenerative diseases, infection and cancer (3). Autophagy marker Light Chain 3 (LC3) was originally identified as a subunit of microtubule-associated proteins 1A and 1B (termed MAP1LC3) (4), and subsequently found to contain similarity to the yeast protein Apg8/Aut7/Cvt5 critical for autophagy (5). Three human LC3 isoforms (LC3A, LC3B, and LC3C) undergo post-translational modifications during autophagy (6-9). Cleavage of LC3 at the carboxy terminus immediately following synthesis yields the cytosolic LC3-I form. During autophagy, LC3-I is converted to LC3-II through lipidation by a ubiquitin-like system involving Atg7 and Atg3 that allows for LC3 to become associated with autophagic vesicles (6-10). The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form LC3-II have been used as indicators of autophagy (11).

  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, 1509-1518.
  3. Levine, B. and Yuan, J. (2005) J. Clin. Invest. 115, 2679-2688.
  4. Mann, S.S. and Hammarback, J.A. (1994) J. Biol. Chem. 269, 11492-11497.
  5. Lang, T. et al. (1998) EMBO J. 17, 3597-3607.
  6. Kabeya, Y. et al. (2000) EMBO J. 19, 5720-5728.
  7. He, H. et al. (2003) J. Biol. Chem. 278, 29278-29287.
  8. Tanida, I. et al. (2004) J. Biol. Chem. 279, 47704-47710.
  9. Wu, J. et al. (2006) Biochem. Biophys. Res. Commun. 339, 437-442.
  10. Ichimura, Y. et al. (2000) Nature 408, 488-492.
  11. Kabeya, Y. et al. (2004) J. Cell Sci. 117, 2805-2812.

Application References

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Companion Products

This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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