Differential Effects of Overexpression of Rab5 and Rab22 on Autophagy in PC12 Cells with or without NGF
Macroautophagy selectively recycles damaged or unneeded proteins and organelles by degradation via targeting to the autophagosome. The following method seeks to identify candidate Rab GTPases that likely modulate autophagy in PC12 cells during nerve growth factor (NGF) starvation. This microscopy-based assay is a single cell-based quantification of the presence of autophagosomes by fluorescently labeled markers in response to the overexpression of Rabs and mutants in the presence or absence of NGF.
Key wordsAutophagy LC3B Rab GTPase GTP-binding protein NGF PC12 cells
The authors’ research program is supported by the NIH/NIGMS grant R01 GM074692 (to G.L.).
- 4.Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, Iemura S, Natsume T, Takehana K, Yamada N, Guan JL, Oshiro N, Mizushima N (2009) Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell 20:1981–1991CrossRefPubMedCentralPubMedGoogle Scholar