Technical Reference #1774

1774.

Biochemical Isolation and Characterization of the Tubulovesicular LC3-positive Autophagosomal Compartment Wentao Gao; Jeong Han Kang; Yong Liao; Wen-Xing Ding; Andrea A. Gambotto; Simon C. Watkins; Yong-Jian Liu; Donna B. Stolz; Xiao-Ming Yin, University of Pittsburgh School of Medicine, Journal of Biological Chemistry, 285(1774), (2010)
Link To Paper

Keywords:
Bacterial transcription; Homeobox; Monoglyceride; Protein export; Radiation inactivation; Respiratory Burst; Autophagosome; Autophagy

Materials & Methods:
Cell Culture and Treatment—HEK-293 and HCT-116 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and other standard supplements. LC3 (specifically LC3B) Atg16L1 Atg5 and Atg9L1 were isolated from human cDNA and cloned to pEGFP or pDsRed/monomer vector (Clontech) at their N terminus. Stable cell lines with GFP-LC3 were created with standard methodology. Transient transfection was conducted using Lipofectamine 2000 (Invitrogen). Cells were treated 48 h later as indicated.

Microscopic Technique
Fluorescence and electron microscopy

Cell Type(s)
HEK-293 and HCT-116 cell