Cell Signaling Technology

Product Pathways - Apoptosis

Atg4B Antibody #5299

Applications Reactivity Sensitivity MW (kDa) Source
W H M R Endogenous 48 Rabbit

Applications Key:  W=Western Blotting
Reactivity Key:  H=Human  M=Mouse  R=Rat
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Atg4B Antibody detects endogenous levels of total Atg4B protein. This antibody detects a band at ~27 kDa of unknown origin.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser372 of human Atg4B protein. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Atg4B Antibody.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with mouse Atg4B (+), using Atg4B Antibody.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Atg4B siRNA I (+), using Atg4B Antibody #5299 (upper) or β-Tubulin (9F3) Rabbit mAb #2128 (lower). The Atg4B Antibody confirms silencing of Atg4B expression, while the β-Tubulin (9F3) Rabbit mAb is used as a loading control.


Background

Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents. Control of autophagy was largely discovered in yeast and involves proteins encoded by a set of autophagy-related genes (Atg) (1). Formation of autophagic vesicles requires a pair of essential ubiquitin-like conjugation systems, Atg12-Atg5 and Atg8-phosphatidylethanolamine (Atg8-PE), which are widely conserved in eukaryotes (2). Numerous mammalian counterparts to yeast Atg proteins have been described, including three Atg8 proteins (GATE-16, GABARAP, and LC3) and four Atg4 homologues (Atg4A/autophagin-2, Atg4B/autophagin-1, Atg4C/autophagin-3, and Atg4D/autophagin-4) (3-5). The cysteine protease Atg4 is pivotal to autophagosome membrane generation and regulation. Atg4 primes the Atg8 homologue for lipidation by cleaving its carboxy terminus and exposing its glycine residue for E1-like enzyme Atg7. The Atg8 homologue is transferred to the E2-like enzyme Atg3 before forming the Atg8-PE conjugate. During later stages of autophagy, Atg4 can reverse this lipidation event by cleaving PE, thereby recycling the Atg8 homologue (6).

While Atg4B displays a broad specificity for Atg8 homologues, it preferentially cleaves LC3 (7-9). Mutation in the corresponding Atg4B gene can be associated with strong inhibition of autophagosome formation. An excess of inactive Atg4B blocks lipidation of Atg8 homologues and inhibits autophagy. This makes Atg4B a potential tool for characterization of the isolation membrane and other autophagy studies (10,11).

  1. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
  2. Ohsumi, Y. (2001) Nat Rev Mol Cell Biol 2, 211-6.
  3. Kabeya, Y. et al. (2000) EMBO J 19, 5720-8.
  4. Kabeya, Y. et al. (2004) J Cell Sci 117, 2805-12.
  5. Mariño, G. et al. (2003) J Biol Chem 278, 3671-8.
  6. Sou, Y.S. et al. (2008) Mol Biol Cell 19, 4762-75.
  7. Hemelaar, J. et al. (2003) J Biol Chem 278, 51841-50.
  8. Kabeya, Y. et al. (2004) J Cell Sci 117, 2805-12.
  9. Tanida, I. et al. (2004) J Biol Chem 279, 36268-76.
  10. Fujita, N. et al. (2008) Mol Biol Cell 19, 4651-9.
  11. Fujita, N. et al. (2009) Autophagy 5, 88-9.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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