Journal: Nature Communications

Article Title: Oxidation of Atg3 and Atg7 mediates inhibition of autophagy

doi: 10.1038/s41467-017-02352-z

Figure Lengend Snippet: Under pro-oxidizing conditions a disulfide forms between the catalytic thiols of Atg3 and Atg7. a siRNA knock-down of Atg7 attenuates intermolecular disulfide oxidation of Atg3, and loss of Atg3 prevents Atg7 disulfide dimerization. b Immunoprecipitation of Atg7 leads co-purification of disulfide-bound mCherry-Atg3. c Overexpressed wild-type mCherryAtg3 and wild-type Atg7 form a disulfide-bound heterodimer in amino-acid-starved cells treated with H 2 O 2 . However, mutation of the catalytic thiol to an alanine on either Atg3 or Atg7 prevents intermolecular disulfide formation

Article Snippet: In some experiments, SMC were transfected for 48 h with a combination of 250 ng of wild-type mCherryAtg3 (Addgene, 54993), C264A mCherryAtg3, wild-type Atg7 (Sino Biological, HG15684) or C572A Atg7 using lipofectamine 3000.

Techniques: Knockdown, Immunoprecipitation, Copurification, Mutagenesis