How to interpret LC3 immunoblotting.
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.
Mizushima
Noboru
N
Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo, Japan. nmizu.phy2@tmd.ac.jp
Yoshimori
Tamotsu
T
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D001343:Autophagy; D045744:Cell Line, Tumor; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008247:Lysosomes / Q000502:physiology; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D016716:PC12 Cells; D010714:Phosphatidylethanolamines / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity
D048868:Adaptor Proteins, Signal Transducing; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D010714:Phosphatidylethanolamines; C483858:phosphatidylethanolamine