Constructs of wild type (WT), dominant-negative (DN) and constitutively active (CA) AMPKα1 in pcDNA3.1 expression vector were generously provided by Prof. David Carling (MRC Clinical Sciences Centre, Imperial College, London, UK). A construct of constitutive active Src (Y529F) in pUSEamp- was purchased from Millipore.
Short hairpin RNA (shRNA) against cSrc was constructed as follows. Two complementary short hairpin siRNA (shRNA) template oligonucleotides, containing 21-nucleotide target sequences of the rat cSrc tyrosine kinase (5′-AAG TAC AAC TTC CAT GGC ACT-3′ , GenBank, AC122515.5), were annealed and ligated into the pScilencer 5.1-H1 Retro vector (Invitrogen, Carlsbad, CA, USA).
Stable transfections of these vectors were performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Individual single cells were isolated and selected with G418 (AMPKα1s and active Src transfected cells, 500 µg/ml) or puromycin (shRNA transfected cells, 5 µg/ml). Phenotypes of the transfected cells were evaluated by AMPK and Src activities (Fig. S1 , S2 ).
Short hairpin RNA (shRNA) against cSrc was constructed as follows. Two complementary short hairpin siRNA (shRNA) template oligonucleotides, containing 21-nucleotide target sequences of the rat cSrc tyrosine kinase (
Stable transfections of these vectors were performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Individual single cells were isolated and selected with G418 (AMPKα1s and active Src transfected cells, 500 µg/ml) or puromycin (shRNA transfected cells, 5 µg/ml). Phenotypes of the transfected cells were evaluated by AMPK and Src activities (