SDS-Page analysis of AICAR action on HCN4 protein phosphorylation. (A) SDS-PAGE gel loaded with HCN4 proteins purified from transfected HEK293F cells under control conditions and after AICAR treatment. The quaternary structure of HCN4 is not completely dissolved in a denaturing gel (Saponaro et al., 2021a; Saponaro et al., 2021b). Therefore, DTT was added to favor the disruption of HCN4 tetramers and the consequent isolation of the monomers. Circled bands were analyzed with nLC-ESI-MS/MS Q Exactive HF. (B) Table of all residues found to be phosphorylated in MS analysis. Source data are available for this figure: SourceData FS4.