A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, The University of Tokyo, Japan
- Department of Molecular Oncology, Institute for Advanced Medical Sciences, Nippon Medical School, Japan
Figures
Figure 1 with 1 supplement
Processed ligand-bound HaloTag from HaloTag-LC3 is a quantifiable readout reflecting autophagic flux.
(a) Immunoblotting of total cell lysates from wild-type and FIP200 knockout (KO) HeLa cells stably expressing monomeric GFP (mGFP)-LC3B or monomeric RFP (mRFP)-LC3B that was in nutrient-rich medium …
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Figure 1—source data 1
Uncropped blot images of Figure 1a, b, c, d and f, and Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/78923/elife-78923-fig1-data1-v2.zip
Figure 1—figure supplement 1
HaloTag-based processing assay can be conducted without a blocking agent.
Wild-type HeLa cells stably expressing HaloTag (Halo)-LC3B, labeled with tetramethylrhodamine (TMR)-conjugated ligand or SF650-conjugated ligand at the indicated concentrations and in the indicated …
Figure 2 with 1 supplement
HaloTag-based reporters can be used to examine autophagic flux by fluorescence imaging.
(a) Fluorescence images of wild-type HeLa cells stably expressing mGFP-LC3B or mRFP-LC3B in nutrient-rich medium or after 2 hr in starvation medium containing 75 nM LysoTracker Deep Red. (b) …
Figure 2—figure supplement 1
HaloTag can be combined with GFP- and/or RFP-based autophagy reporters.
(a) Fluorescence images of wild-type HeLa cells stably expressing lysosomal marker LAMP1-mRuby3 and HaloTag (Halo)-LC3B in nutrient-rich medium or after 6 hr in starvation medium containing 200 nM …
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Figure 2—figure supplement 1—source data 1
Uncropped blot images of Figure 2—figure supplement 1e.
- https://cdn.elifesciences.org/articles/78923/elife-78923-fig2-figsupp1-data1-v2.zip
Figure 3 with 1 supplement
HaloTag-based reporters can be adapted to monitor selective autophagy.
(a) Immunoblotting and in-gel fluorescence detection of wild-type and FIP200 knockout (KO) HeLa cells stably expressing HaloTag (Halo)-mGFP-KDEL pulse-labeled for 20 min with 100 nM …
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Figure 3—source data 1
Uncropped blot images of Figure 3a, c and g.
- https://cdn.elifesciences.org/articles/78923/elife-78923-fig3-data1-v2.zip
Figure 3—figure supplement 1
Halo-GFP-KDEL and pSu9-Halo-GFP localize to endoplasmic reticulum (ER) and mitochondria, respectively.
(a) Fluorescence images of wild-type HeLa cells stably expressing HaloTag (Halo)-mGFP-KDEL and ER marker mRuby3-cytochrome b5 in nutrient-rich medium containing 200 nM SF650-conjugated ligand. (b) …
Figure 4 with 1 supplement
Bulk nonselective autophagic flux can be detected with HaloTag-GFP.
(a,b) Immunoblotting and in-gel fluorescence detection of total cell lysates from wild-type, FIP200 knockout (KO), ATG3 KO, and ATG5 KO HeLa cells stably expressing HaloTag (Halo)-LC3B (a) or …
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Figure 4—source data 1
Uncropped blot images of Figure 4a and b.
- https://cdn.elifesciences.org/articles/78923/elife-78923-fig4-data1-v2.zip
Figure 4—figure supplement 1
Bulk nonselective autophagic flux can be detected with Halo-GFP.
(a) Fluorescence images of wild-type HeLa cells stably expressing lysosomal marker LAMP1-mRuby3 and HaloTag (Halo)-mGFP under nutrient-rich conditions or after 6 hr under starvation conditions in …
Author response image 1
HeLa cells stably expressing pSu9-Halo-GFP in growing medium with 200 nM SF650 ligand.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/78923/elife-78923-mdarchecklist1-v2.docx
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Supplementary file 1
Sequences of the primers used for plasmid construction.
- https://cdn.elifesciences.org/articles/78923/elife-78923-supp1-v2.xlsx
