LC3/LC3B - measuring autophagosome formation and autophagic flux

Fri, 05/01/2015 - 13:12

Microtubule-associated protein-1 light chain 3 (LC3/LC3B) is a ubiquitin-like protein involved in the formation of the autophagosome. It is homologous to the yeast Atg8 protein. Autophagosomes are important for the degradation and recycling of intracellular cargo such as misfolded proteins or damaged organelles. Upon induction of autophagy, LC3 is conjugated to the lipid phosphatidylethanolamine (PE) by the Atg12-Atg5-Atg16 protein complex. This lipidation is essential for the localization of LC3 to the assembling autophagosome where it recruits the core autophagy machinery. LC3 mediates membrane expansion in order to selectively engulf cargo and deliver it to the lysosome for degradation. Autophagy receptors, such as p62/SQSTM1, recognize specific ubiquitinated cargo. They also contain LC3-interacting regions (LIR) which allow them to deliver cargo to the autophagosome by binding to LC3. The LC3 protein contained within the autophagosome is degraded along with associated cargo. This turnover of LC3 offers an effective way of monitoring the induction and rate of autophagy within cells by immunoblotting or immunofluorescence. The amount of LC3B conjugated to PE (LC3B-PE) is correlated to the number of autophagosomes within a cell and serves as a general marker for autophagosome formation. LC3B antibodies are used for immunofluorescence to visualize the formation and degradation of autophagosomes and to look at colocalization with markers for various cargo. Similarly LC3B antibodies can be used for immunoblotting to compare the relative amounts of cytosolic LC3B and membrane bound LC3B-PE in order to monitor autophagic flux.

This method of measuring autophagic flux using the LC3B antibody for immunoblotting was employed by Jang et al. to examine the mechanism of hypoglycemia-induced neuronal cell death (1). They found inhibition of autophagic flux after glucose reintroduction after periods of hypoglycemia can lead to an increase in caspase-3 activity and cell death. Researchers at the Mayo Clinic demonstrated a new role for Dynamin 2 in the autolysosomal breakdown of cellular lipid droplets (2). This study used LC3/LC3B antibodies in immunofluorescence to show accumulation of autophagosomes and block of autophagy following depletion of Dynamin 2. The Rubinsztein group from University of Cambridge published research in Cell examining membrane sources during autophagosome formation (3). They showed VAMP3 mediates the fusion of membrane vesicles derived from recycling endosomes to provide an essential source of membrane for autophagosome formation. They used the LC3 antibody to show VAMP3 depletion prevented the formation of LC3-PE indicating a reduction in autophagosomes. Proenca et al. monitored autophagic flux using the LC3 antibody in a mouse model of Huntington's disease (4). Their study showed induction of Atg4 dependent autophagic flux alleviates protein aggregate accumulation and disease progression. The Avantaggiati group at Georgetown University showed degradation of mutant p53 protein through autophagy may have important implications in tumor evolution (5). They used the LC3 antibody to monitor the induction or inhibition of autophagy and the effect on mutant p53 levels.

Novus Biologicals offers LC3 reagents for your research needs including:


  1. 24124562
  2. 24145164
  3. 24034251
  4. 23861892
  5. 23466706

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