Following autophagy step by step | BMC Biology | Full Text
2011年6月2日 · A tandem fusion of mCherry and GFP is fused to LC3B (one of the several members of the mammalian LC3 family) to make a pH-sensitive sensor that is used to monitor autophagy in live cells. The GFP tag is acid-sensitive while the mCherry tag is acid-insensitive.
Monitoring Autophagy Flux and Activity: Principles and ...
2020年8月26日 · Monitoring GFP-LC3 and RFP-GFP-LC3 images traces the dynamic membrane transition from autophagosome to autolysosome. Meanwhile, a fluorescent protein, Keima, has been used for monitoring the transition of an autophagy substrate during autophagosomal maturation into autolysosomes.
Monitoring Autophagy with GFP-LC3 Reporter - PubMed
2023年10月28日 · In this chapter, we will detail step-by-step the GFP-LC3 reporter protocol we have adapted in our lab. This protocol begins with the generation of lentivirus expressing GFP-LC3. Then, the cells are transduced with titrated virus.
Dissecting the dynamic turnover of GFP-LC3 in the autolysosome
Here we found that lysosomal acidity is an important regulating factor for the step-wise degradation of GFP-LC3, in which the free GFP fragments are first generated but accumulate only when the lysosomal acidity is moderate, such as during rapamycin treatment.
Methods for Measuring Autophagy in Mice - PMC - National …
2017年6月8日 · To avoid these artifacts, GFP-LC3 transgenic mice samples have to be compared with non-transgenic control littermates, with GFP fluorescence detected using only narrow-band pass GFP or FITC filter sets.
Fluorescent Biosensors for Measuring Autophagic Flux
LC3 is a commonly used marker for measuring autophagic flux. mRFP-GFP-LC3 functions as an autophagy bionsensor via the same mechanism as Rosella: GFP is quenched in acidic environments, while mRFP is relatively stable and fluorescences even at …
Monitoring Autophagy with GFP-LC3 Reporter | SpringerLink
2023年10月28日 · Among these methodologies, GFP-LC3 reporter has been popularly used in the literature. In this chapter, we will detail step-by-step the GFP-LC3 reporter protocol we have adapted in our lab. This protocol begins with the generation of lentivirus expressing GFP-LC3.
Autophagosome immunoisolation from GFP-LC3B mouse tissue
2018年10月30日 · We describe a protocol for rapid and efficient enrichment of autophagosomes from various tissues of the GFP-LC3 mouse. In order to increase the number of autophagosomes, we block autophagy flux in the GFP-LC3 mouse tissue with a single intraperitoneal injection of leupeptin 4–5 h before tissue harvesting.
Transgenic expression of GFP-LC3 perturbs autophagy in exocrine ...
Unexpectedly, GFP-LC3 expression markedly increased endogenous LC3-II level in pancreas, caused by downregulation of ATG4B, the protease that deconjugates/delipidates LC3-II. By contrast, GFP-LC3 expression had lesser or no effect on autophagy in liver, lung and spleen.
GFP-LC3 High-content Assay for Screening Autophagy Modulators
This assay takes advantage of the distinctive pattern of cellular GFP-LC3 upon autophagy induction. Under nutrient-rich conditions, the level of autophagy is low and GFP-LC3 displays a diffuse pattern. However, during cellular stress, the process of …