Cell-based kinase assay. PKC activator PMA, PKCa inhibitor Gö6976 and Akt activator IGF-1 and inhibitor MK-2206 were used to activate or block signaling pathways in C2 cells. For targeted MS analysis, phosphopeptides were enriched by Myc-tag and TiO2-based enrichment.
Immunoblot analysis of PKCα and Akt activities in C2 cells following pharmacologic interventions as indicated in 4C. Pan- and phospho-specific antibodies were used to detect total protein amounts and phosphoisoforms. GAPDH was used as loading control.
Targeted MS data of hFLNc phosphopeptides comprising the phosphorylation sites pS2233, pS2233/pS2236 and pS2236. MS data were quantified using Skyline and normalized to an internal phosphopeptide standard and the mock-treated control (DMSO). Intensities of phosphopeptides distinctive for a specific phosphorylation site in hFLNc d18-21 WT were added up per experiment and represented as normalized mean log2 ratio (treatment/control) ± SEM (n=4; *, p ≤ 0.05; **, p ≤ 0.01)