In vitro kinase assay with human filamin C domain 18-21 recombinantly expressed in E.coli, purified by Ni2+-NTA beads and incubated with PKC alpha, Akt or a combination of both to identify kinase specific phosphorylation sites.

Recombinantly expressed and purified mouse and human FLNc d18-21 WT was dialyzed overnight at 4°C in dialysis buffer (1 mM DTT, 100 mM KCl, 20 mM HEPES pH 7.4, 10 mM MgCl2). For MS-coupled kinase assays, H2O was added to 100 µg protein to a total volume of 200 µl and mixed with 10 x kinase buffer (NEB, Frankfurt, Germany). The assay was started by adding 200 ng Akt (Proteinkinase, Kassel, Germany) and/or 200 ng PKC alpha (Sigma-Aldrich) in the presence of 1x PKC lipid activator (Merck Millipore, Darmstadt, Germany). The reaction was carried out for 20 min at 30°C and 200 rpm. One tenth of each of the three independent replicates was used for gel-based analyses. The remaining sample was diluted 1:4 (v/v) with 50 mM ammonium bicarbonate and subjected to in-solution digestion using sequencing grade trypsin (1:50) (Promega) for 3.5 h at 42°C and 200 rpm on a thermoshaker. Single protein digests were acidified with TFA [final concentration 1% (v/v)], subjected to TiO2-based phosphopeptide enrichment, and analyzed by LC-MS/MS using MSA, HCD and ETD fragmentation.