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How to Interpret LC3 Immunoblotting
Open accessAbstract
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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“LC3, which is a post-translational modifier, is required for autophagosome formation (Mizushima and Yoshimori, 2007).”
From Inducible Rpt3, a Proteasome Component, Knockout in Adult Skeletal Muscle Results in Muscle Atrophy (2020) · cites this paper as Mizushima and Yoshimori, 2007
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“In neurons ATG9 localizes to axons and presynaptic sites, and requires active transport by the kinesin motor KIF1A to direct its localization into distal neurites [30,537].”
From Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) 1 (2021) · cites this paper as [30]
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LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.
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Self-citationThe role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress
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Self-citationLC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation
Kabeya, Y., Mizushima, N., Yamamoto, A., Oshitani-Okamoto, S., Ohsumi, Y., & Yoshimori, T. (2004) · Journal of Cell Science · doi:10.1242/jcs.01131 · Cited by 1,394
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Self-citationLysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy
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Methods for monitoring autophagy
Mizushima, N. (2004) · The International Journal of Biochemistry & Cell Biology · doi:10.1016/j.biocel.2004.02.005 · Cited by 899
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Wang, L., Xu, W., Zhou, Q., Xu, B., Sheng, Y., Sun, M., Chen, H., Wang, Y., Ding, G., & Duan, Y. (2020) · Toxicological Sciences · doi:10.1093/toxsci/kfaa116 · Cited by 3
Joo, H.-W., Song, Y.-S., Park, I.-H., et al. (2020) · Canadian Journal of Gastroenterology and Hepatology · doi:10.1155/2020/2156829 · Cited by 3
Selvan, S. R., Brichetti, J. A., Thurber, D. B., Botting, G. M., & Bertenshaw, G. P. (2021) · Cancer Biotherapy and Radiopharmaceuticals · doi:10.1089/cbr.2021.0283 · Cited by 3
Lyon, A., Tripathi, R. P., Meeks, C., et al. (2023) · Cancers · doi:10.3390/cancers15030954 · Cited by 3
Oxidative Stress and Dysfunction of the Intracellular Proteolytic Machinery
Homma, T., & Fujii, J. (2019) · Elsevier eBooks · doi:10.1016/b978-0-12-814466-4.00005-7 · Cited by 3
Hao, Y., Zheng, C., Qiang, H., et al. (2019) · ChemNanoMat · doi:10.1002/cnma.201900572 · Cited by 3
Crosstalk between Autophagy and Apoptosis Modulates T-2 Toxin-Induced Toxicity in Liver CellsPreprint
Wu, J., Zhou, Y., Yuan, Z., et al. (2018) · Preprints.org · doi:10.20944/preprints201810.0034.v1 · Cited by 3
Sun, F., Tong, Y., Zhang, C., Yang, S., Liu, Y., Ji, J., Yu, Z., & Zhao, Y. (2023) · Research Square · doi:10.21203/rs.3.rs-2520455/v1 · Cited by 3
TDP-43 pathology links innate and adaptive immunity in amyotrophic lateral sclerosisPreprint
Evangelista, B., Ragusa, J. V., Pellegrino, K., Wu, Y., Quiroga, I. Y., Cahalan, S. R., Arooji, O. K., Madren, J. A., Schroeter, S., Cozzarin, J., et al. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.01.07.574541 · Cited by 4
Urban, N. D., Lacy, S. M., Pelt, K. M. V., et al. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.01.14.633073 · Cited by 3
Biallelic variants in SREBF2 cause autosomal recessive spastic paraplegia.
Wei, Q., Fan, W., Li, H.-F., Wanga, P.-S., Xu, M., Dong, H.-L., Yu, H., Lyu, J., Luo, W.-J., Chen, D.-F., et al. (2025) · Journal of genetics and genomics = Yi chuan xue bao · doi:10.1016/j.jgg.2025.01.004 · Cited by 2
Metadata
- Authors
- Mizushima, N., & Yoshimori, T.
- Affiliations
- Tokyo Medical and Dental University (JP)
- Venue
- Autophagy
- Publisher
- Informa UK Limited
- Published
- 2007-11-26
- Volume
- 3
- Issue
- 6
- Pages
- 542-545
- ISSN
- 1554-8627 (ISSN-L)
- 1554-8635
- OpenAlex
- W1963521777
- Semantic Scholar
- ab5143ed104a787ff2e14d915df9a12ce5f5871a
- Cited by
- 2,595
- References
- 21
- Citations by year
- 2026: 62 · 2025: 114 · 2024: 152 · 2023: 141 · 2022: 176 · 2021: 164 · 2020: 173 · 2019: 155 · 2018: 177 · 2017: 162 · 2016: 167 · 2015: 179 · 2014: 137 · 2013: 165 · 2012: 120
- FWCI
- 52.76
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by-nc
- Locations
- Autophagy (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- Funders
- Kato Memorial Bioscience Foundation
- Toray Science Foundation
- Japan Society for the Promotion of Science
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Calcium signaling and nucleotide metabolism
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Keywords
- LC3, autophagic flux
- MeSH terms
- Animals, Autophagy, Humans, Lysosomes, Microtubule-Associated Proteins, Phosphatidylethanolamines, Sensitivity and Specificity, Immunoblotting, PC12 Cells, Cell Line, Tumor, Adaptor Proteins, Signal Transducing, Rats
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