In vitro kinase assay coupled to phosphorylation-dependent mobility shift analysis. Reactions were performed using recombinant hFLNc d18-21 WT in the presence (+) or absence (-) of ATP, Akt and/or PKCα as indicated. Immunoblot analysis performed with an antibody directed against the EEF-tag that was fused to the carboxy-terminus of hFLNc d18-21 WT revealed multiple shifted bands, indicating kinase-dependent phosphorylation events. h, human; WT, wild-type
In vitro kinase assay coupled to quantitative MS analysis for site determination. Reactions were performed as described in figure 4A. MS data for each kinase were quantified using Skyline and are available in this Panorama project. Intensities of phosphopeptides distinctive for a specific phosphorylation site in hFLNc d18-21 WT were added up per experiment and represented as normalized mean ± SEM (n=3).