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Autophagy

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The 2016 Nobel Prize in Physiology or Medicine was awarded to Dr. Yoshinori Ohsumi “for his discoveries of mechanisms for autophagy” [1]. Genetic resources for autophagy study are available. Looking forward to receiving your request.

Resarch Tools of Autophagy

A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag.

Yim, W.W., Yamamoto, H., Mizushima, N.
Elife 11: e78923 (2022). PubMed PMID 35938926.

Catalog no. Name of resource Description
RDB19962 pMRX-IB-pSu9-HaloTag7-mGFP Retroviral vector for stable expression of pSu9-HaloTag7-mGFP to measure mitophagy activity.
RDB19963 pMRX-IB-HaloTag7-mGFP-KDEL Retroviral vector for stable expression of HaloTag7-mGFP-KDEL to measure ER-phagy activity.
RDB19964 pMRX-IB-HaloTag7-mGFP Retroviral vector for stable expression of HaloTag7-mGFP to measure autophagic flux.
RDB19965 pMRX-No-HaloTag7-mGFP-LC3-mRFP Retroviral vector for stable expression of HaloTag7-mGFP-LC3-mRFP to measure autophagic flux.
RDB19966 pMRX-No-HaloTag7-mGFP-LC3 Retroviral vector for stable expression of HaloTag7-mGFP-LC3 to measure autophagic flux.
RDB19967 pMRX-No-HaloTag7-LC3 Retroviral vector for stable expression of HaloTag7-LC3 to measure autophagic flux.
RDB19968 pMRX-IP-HaloTag7-LC3 Retroviral vector for stable expression of HaloTag7-LC3 to measure autophagic flux.

Intrinsically Disordered Protein TEX264 Mediates ER-phagy.

Chino, H., Hatta, T., Natsume, T., Mizushima, N.
Mol. Cell 74 (5): 909-921.e6 (2019). PubMed PMID 31006538.

Catalog no. Name of resource Description
RDB19767 pCW57-CMV-ssRFP-GFP-KDEL Lentiviral vector for inducible expression of ER-phagy marker ssRFP-GFP-KDEL.

An Autophagic Flux Probe that Releases an Internal Control.

Kaizuka, T., Morishita, H., Hama, Y., Tsukamoto, S., Matsui, T., Toyota, Y., Kodama, A., Ishihara, T., Mizushima, T., Mizushima, N.
Mol. Cell 64 (4): 835-849 (2016). PubMed PMID 27818143.

Catalog no. Name of resource Description
RDB14600 pMRX-IP-GFP-LC3-RFP-LC3 delta G Fluorescent probe to evaluate autophagic flux by the rat LC3 fused with GFP and RFP.
RDB14601 pMRX-IP-GFP-LC3-RFP Fluorescent probe to evaluate autophagic flux by the rat LC3 fused with GFP and RFP.
RDB19804 pcDNA3-GFP-LC3-RFP-LC3 deltaG An autophagic flux probe, expression vector of GFP-rat LC3-RFP-rat LC3 deletion G.
  • In order to visualize autophagosome and observe autophagy, MAP1LC3 (LC3) fused with fluorescent proteins as a probe are used. However, the fluorescence of the probe attenuates during autophagy progresses and it is not suitable for quantitative assay of autophagy.
  • Dr. Noboru Mizushima and his colleagues at the University of Tokyo developed a new probe that can expresses GFP-LC3 and RFP-LC3 delta G (lacking glycine (G) at the LC 3 end) simultaneously at equimolar amounts. As autophagy progresses, the fluorescence of GFP-LC3 decays whereas that of RFP-LC3 delta G remains in the cytoplasm as an internal standard. Therefor, the activity of autophagy can be quantified evaluated by the fluorescence intensity ratio of GFP/RFP.
  • Using this probe, autophagic flux was measured in mouse and zebrafish embryos and tissues as well as cultured cells. This is also suitable for analysis of large number of samples.

Establishment of a novel fluorescence-based method to evaluate chaperone-mediated autophagy in a single neuron.

Seki, T., Yoshino, K.I., Tanaka, S., Dohi, E., Onji, T., Yamamoto, K., Hide, I., Paulson, H.L., Saito, N., Sakai, N.
PLoS One 7 (2): e31232 (2012). PubMed PMID 22363588.

Catalog no. Name of resource Description
RDB15088 pcDNA5-GAPDH-HaloTag Expression vector for the visualization of chaperone-mediated autophagy (CMA).
  • The GAPDH-HT indicator is a chaperone-mediated autophagy (CMA) marker consisting of HaloTag and GAPDH which is a typical substrate of CMA. The CMA activity in the cells can be evaluated by using GAPDH-HT indicator and an appropriate under the fluorescent-labeled HaloTag ligand.

References and Related Articles:

  • Sato, M. et al. Fluorescent-based evaluation of chaperone-mediated autophagy and microautophagy activities in cultured cells. Genes Cells 21 (8): 861-873, 2016. PubMed PMID: 27377049

A conditional proteomics approach to identify proteins involved in zinc homeostasis.

Miki, T., Awa, M., Nishikawa, Y., Kiyonaka, S., Wakabayashi, M., Ishihama, Y., Hamachi, I.
Nat. Methods 13 (11): 931-937 (2016). PubMed PMID 27617391.

Catalog no. Name of resource Description
RDB15159 mCherry-LC3B Autophagosome marker. Expression vector of rat Microtubule-associated proteins 1A/1B light chain 3B (LC3B), mCherry tag.

The nucleolar protein nucleophosmin is essential for autophagy induced by inhibiting Pol I transcription.

Katagiri, N., Kuroda, T., Kishimoto, H., Hayashi, Y., Kumazawa, T., Kimura, K.
Sci. Rep. 5: 8903 (2015). PubMed PMID 25754892.

Catalog no. Name of resource Description
RDB13435 pEGFP-C1-hLC3B Indicator of autophagy. Expression clone of human LC3B in mammalian cells.

Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane.

Yoshii, S.R., Kishi, C., Ishihara, N., Mizushima, N. J. Biol. Chem. 286 (22): 19630-19640 (2011). PubMed PMID 21454557.

Catalog no. Name of resource Description
RDB19786 pMXs-IP-GFP-Omp25 Retroviral vector for stable expression of rat Omp25 with N-terminal EGFP.

Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins.

Itakura, E., Mizushima, N. Autophagy 6 (6): 764-776 (2010). PubMed PMID 20639694.

Catalog no. Name of resource Description
RDB19630 pMXs-IP-GFP-WIPI1 Retroviral vector for stable expression of human WIPI1 with N-terminal EGFP.
RDB19768 pMXs-IP spGFP-ERGIC53 Retroviral vector for stable expression of human ERGIC-53 with N-terminal GFP.

FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells.

Hara, T., Takamura, A., Kishi, C., Iemura, S., Natsume, T., Guan, J.L., Mizushima, N. J. Cell Biol. 181 (3): 497-510 (2008). PubMed PMID 18443221.

Catalog no. Name of resource Description
RDB19634 pMXs-IP-EGFP-mAtg5 Retroviral vector for stable expression of mouse Atg5 with C-terminal EGFP.
RDB19798 pMXs-IP-EGFP-LC3 Retroviral vector for stable expression of rat LC3 with N-terminal EGFP.

List of gene resources

References

  1. The 2016 Nobel Prize in Physiology or Medicine
  2. Tsukada, M., FEBS Lett. 333 (1-2): 169-174 (1993).
  3. Kabeya, Y., Biochem. Biophys. Res. Commun. 356 (2): 405-410 (2007).
  4. Mizushima, N., Annu. Rev. Cell Dev. Biol. 27: 107-132 (2011).
  5. KEGG Pathway – Autophagy – animal
  6. KEGG Pathway – Autophagy – yeast
  7. KEGG Pathway – Autophagy – other

(GSB0021e, T.M. 2014.10.13)

2023.08.20



 

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