Fig. 1.

Phosphoproteomic screening to identify AMPK substrates. a The phosphoproteomics workflow. AMPKα1/α2–double knockout (DKO) HEK293A-derived cells were generated using the CRISPR-Cas9 genome-editing technology. These cells were used as controls to compare with parental (WT) cells with intact AMPK. SILAC-based quantitative phosphoproteomic analysis was performed to identify AMPK substrates. GFP green fluorescent protein, RP reverse phase, LC-MS liquid chromatograph-mass spectrometry, A769662 an AMPK activator. b Validation of AMPKα1/α2-DKO cells using anti-AMPK antibodies and antibodies recognizing known AMPK phospho-substrates. WT AMPK cells were used as a control. c Time course of the effect of treatment with the AMPK activator A769662 (100 μM) on WT HEK293A cells. Western blotting was conducted using antibodies as indicated. A treatment period of 24 h was selected because it led to maximal AMPK kinase activity as indicated by AMPKα phosphorylation at the Thr-172 site