Fig. 1. Protein microarray screen for the identification of AMPK substrates.

a Schematic representation of the ProtoArray based screen with approximately 9000 human proteins using AMPK (see also Supplementary Data 1). b Details of two sub-arrays incubated with or without AMPK with marked substrates are shown. c GST-CDK16, Cyclin Y-His₆ and GST were incubated in the presence of [γ-³²P]-ATP with AMPK. Phosphorylation was determined by autoradiography (³²P, top). Proteins were visualized by Coomassie blue staining (CB, bottom; n = 2). d HeLa cells were transfected with vectors expressing GFP-CDK16 and Cyclin Y-Flag and treated for 1 h with 0.5 mM AICAR/50 µM A769662 (A769) as indicated. Cyclin Y-Flag was immunoprecipitated with Flag antibodies (IP) and immunoblotted against CDK16 and Cyclin Y or used for in vitro kinase assays with myeloid basic protein (MBP) as substrate. Autoradiographs (³²P) and Coomassie blue staining (CB) of MBP are displayed. Whole cell lysates (WCL) were immunoblotted with the indicated antibodies (n = 3). e Quantification of CDK16 co-immunoprecipitated with Cyclin Y. Statistical significance was measured via unpaired and two-tailed Student’s t-tests and is presented as follows: **p < 0.01, and ***p < 0.001. All error bars indicate SD (n = 3; Cyclin Y + AICAR/A769 vs. Cyclin Y/CDK16: t = 8.719, df = 4; Cyclin Y/CDK16 vs. Cyclin Y/CDK16 + AICAR/A769: t = 5.595, df = 4). n biological independent replicate. SD standard deviation. Source data are provided as a Source Data file.