How to interpret LC3 immunoblotting

Autophagy. Nov-Dec 2007;3(6):542-5. doi: 10.4161/auto.4600. Epub 2007 Jun 19.

Abstract

Microtubule-associated protein light chain 3 (LC3) is now widely used to monitor autophagy. One approach is to detect LC3 conversion (LC3-I to LC3-II) by immunoblot analysis because the amount of LC3-II is clearly correlated with the number of autophagosomes. However, LC3-II itself is degraded by autophagy, making interpretation of the results of LC3 immunoblotting problematic. Furthermore, the amount of LC3 at a certain time point does not indicate autophagic flux, and therefore, it is important to measure the amount of LC3-II delivered to lysosomes by comparing LC3-II levels in the presence and absence of lysosomal protease inhibitors. Another problem with this method is that LC3-II tends to be much more sensitive to be detected by immunoblotting than LC3-I. Accordingly, simple comparison of LC3-I and LC3-II, or summation of LC3-I and LC3-II for ratio determinations, may not be appropriate, and rather, the amount of LC3-II can be compared between samples.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Animals
  • Autophagy*
  • Cell Line, Tumor
  • Humans
  • Immunoblotting / methods*
  • Lysosomes / physiology
  • Microtubule-Associated Proteins / metabolism*
  • PC12 Cells
  • Phosphatidylethanolamines / metabolism
  • Rats
  • Sensitivity and Specificity

Substances

  • Adaptor Proteins, Signal Transducing
  • Microtubule-Associated Proteins
  • Phosphatidylethanolamines
  • light chain 3, human
  • phosphatidylethanolamine