How to Interpret LC3 Immunoblotting

By N. Mizushima, T. Yoshimori. Autophagy, 2007

2,548 citations recorded by the source corpus.

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.

Subfields: Cell Biology, Epidemiology, Physiology

View Paper: https://www.semanticscholar.org/paper/ab5143ed104a787ff2e14d915df9a12ce5f5871a

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Citations in the corpus

Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains (2009, methodology): “Third, we used bafilomycin A1, a specific inhibitor of vacuolar H -ATPases and a blocker of autophagosome-lysosome fusion (20, 31).”
The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice. (2008, background): “While LC3-I is present in the cytosol, the lipidated LC3-II form is associated mainly with autophagosomal membranes, serving as a good indicator of autophagosomes (32, 33).”
Autophagy: process and function. (2007, background): “One current problem is that this degradation step is rather difficult to measure, although some methods to monitor autophagy flux have been proposed (Tanida et al. 2005; Klionsky et al. 2007; Mizushima and Yoshimori 2007).”
Autophagy-Dependent Ferroptosis: Machinery and Regulation (2020, background): “At the same time, MAP1LC3-II can be degraded in a lysosome-dependent manner (Mizushima and Yoshimori, 2007; Pankiv et al., 2007).”
Endogenous HMGB1 regulates autophagy (2010, background): “(E) Analysis of p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.005 and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3.”
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (2012, positive): “tion independently of traditional autophagy, in a noncanonical autophagic process termed LC3-associated phagocytosis.(1,26,1448) LAP occurs upon engulfment of particles that engage a recep-”
Pathogenic Lysosomal Depletion in Parkinson's Disease (2010, background): “Because LC3II is normally degraded by lysosomal proteases once the AP fuses with a lysosome to form an autophagolysosome (Mizushima and Yoshimori, 2007), the accumulation of LC3II protein observed following MPTP intoxication indicates that, in addition to any potential induction of new AP…”
Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. (2008, background): “We also monitored the expression level of autophagy substrate p62, another indicator of autophagic degradation (Bjørkøy et al., 2005; Mizushima and Yoshimori, 2007).”
The dynamic nature of autophagy in cancer. (2011, background): “The lipidated (LC3-II) form can be detected as a faster-migrating band by immunoblotting (Mizushima and Yoshimori 2007).”
Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria. (2010, positive): “Amount of LC3-II was normalized as LC3-I/(LC3-I+LC3-II), described as the most accurate quantitative method (Mizushima and Yoshimori, 2007).”