Generation of AMPK Mutant Cell Lines

pCDNA3 plasmid harbouring Myc-AMPKα1(WT), Myc-AMPKα1(S173C) or Myc-AMPKα1(S173D) [39 (link), 40 (link)], kindly given by Dr. Dietbert Neumann (Maastricht University, The Netherlands), were used to generate populations of C3A cells which stably express mutated forms of the AMPKα1(S173) residue. C3A cells were transfected by electroporation, as previously described [37 (link)]. After 24 h, the antibiotic Geneticin (500 μg/ml) (Invitrogen, Thermo Fisher Scientific) was added to the media in order to select the transfected cells. Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells.