How to Interpret LC3 Immunoblotting
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Overview
Technical review demonstrates critical pitfalls in LC3 immunoblotting for monitoring autophagic flux, highlighting the need to assess lysosomal turnover rather than static protein ratios.
Key Points
- To outline best practices and address analytical pitfalls when interpreting LC3 immunoblotting to monitor autophagy and autophagic flux.
- Evaluation of immunoblotting techniques that detect the conversion of LC3-I to LC3-II as an indicator of autophagosome abundance.
- Methodological assessment of measuring LC3-II lysosomal turnover using lysosomal protease inhibitors versus static ratio comparisons.
- Static quantification of LC3-II levels does not indicate autophagic flux because LC3-II is degraded within lysosomes during the autophagic process.
- Differential immunoblotting sensitivity between LC3-I and LC3-II makes ratio determinations or summations inappropriate, requiring comparison of LC3-II levels between samples in the presence and absence of lysosomal protease inhibitors.
Cite This Study
Mizushima et al. (2007) studied this question.