Figure S3. 

A, Radioactive in vitro kinase assays. Recombinant wildtype and phosphosite mutants of mouse and human FLNc Ig-like domains 23-24 (d23-24) were treated with [γ-33P]ATP in the absence of PKCα and analysed by SDS-PAGE followed by autoradiography or Coomassie staining. S2625 of mFLNC d23-24 was replaced by A or D; S2623/S2624 of hFLNC d23-24 by AA or DD. As a control, PKCα was incubated in [γ-33P]ATP-containing kinase buffer without hFLNc d23-24 (B). WT, wildtype; A, alanine; D, aspartate

B, Abundance of recombinant mouse and human FLNc d23-24 wildtype and phosphosite mutants in mass spectrometry-based in vitro kinase assays. Shown are the mean values of the total peptide intensity measured for each of the FLNc d23-24 constructs (see Figure S2B). Data derived from three independent experiments per construct. Error bars represent the SEM. WT, wildtype; A, alanine, D, aspartate; n.s., not significant