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How to Interpret LC3 Immunoblotting

Noboru Mizushima and Tamotsu Yoshimori

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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.

Authors

Noboru Mizushima

Department of Physiology and Cell Biology; Tokyo Medical and Dental University; Tokyo Japan

Tamotsu Yoshimori

Department of Cellular Regulation; Research Institute for Microbial Diseases; Osaka University; Suita, Osaka Japan

This is an open-access article

 Download PDF

If the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.