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      How to interpret LC3 immunoblotting.

      Autophagy

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

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          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.

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          Journal
          17611390
          10.4161/auto.4600

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