ArticleeLife2022
A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag.
Article in eLife, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 87 papers.
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Who cites it
87 citing papers in PubMed, 140 citations in OpenAlex.
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- Discerning protein pools by selective staining with self-labeling tags.FEBS letters · 2026Review
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- Activation of pro-survival autophagy by a small molecule promoting p62 oligomerization.The Journal of biological chemistry · 2026Article
- Regulatory mechanisms of post-translational modifications on autophagic flux in neurons following ischemic stroke.Molecular biology reports · 2026Review
- Alternol-Induced Oxidative Modification of SQSTM1/p62 Is Associated with Nrf2 Signaling and Autophagy-Related Responses in Prostate Cancer Cells.Antioxidants (Basel, Switzerland) · 2026Article
- Metformin Redirects Autophagy from Bulk Turnover to Mitochondrial Clearance.bioRxiv : the preprint server for biology · 2026Article
- Evaluating the involvement of autolysosomes in the nuclear translocation of fluorescent proteins.FEBS open bio · 2026Article
- Quantitative analysis of autophagic flux reveals radiation-induced activation of SQSTM1-mediated degradation of protein aggregates and ER-phagy.Journal of radiation research · 2026Article
- Bi-allelic ATG12 variants impair autophagy and cause a neurodevelopmental disorder.American journal of human genetics · 2026Article
- Cutting edge: ESCRT-mediated phagophore closure in mammals.The Biochemical journal · 2026Review
- Disrupted autophagy overactivates TBK1 and results in mitotic defects promoting chromosomal instability.Autophagy · 2026Article
- Article
- TOLLIP targets GSDME-NT-carrying endocytic vesicles for autophagy to regulate pyroptosis and chemotherapy efficacy.Nature cell biology · 2026Article
- Control of lysosome function by the GTPase-activating protein TBC1D9B and its binding partner TMEM55B.Nature communications · 2026Article
- A Rab1 interactome illuminates a dual role in autophagy and membrane trafficking.The Journal of cell biology · 2026Article
27 more citing papers are in PubMed but not listed here.
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Authors and funding
3 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Monitoring autophagic flux is necessary for most autophagy studies. The autophagic flux assays currently available for mammalian cells are generally complicated and do not yield highly quantitative results. Yeast autophagic flux is routinely monitored with the green fluorescence protein (GFP)-based processing assay, whereby the amount of GFP proteolytically released from GFP-containing reporters (e.g. GFP-Atg8), detected by immunoblotting, reflects autophagic flux. However, this simple and effective assay is typically inapplicable to mammalian cells because GFP is efficiently degraded in lysosomes while the more proteolytically resistant red fluorescent protein (RFP) accumulates in lysosomes under basal conditions. Here, we report a HaloTag (Halo)-based reporter processing assay to monitor mammalian autophagic flux. We found that Halo is sensitive to lysosomal proteolysis but becomes resistant upon ligand binding. When delivered into lysosomes by autophagy, pulse-labeled Halo-based reporters (e.g. Halo-LC3 and Halo-GFP) are proteolytically processed to generate Halo
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.