Calmodulin

For analytical purposes, 0.2 mg/ml obscurin RhoGEF fragments (5–20 μM, depending on the fragment used) were phosphorylated by 200 U PKA (Sigma-Aldrich, cat. no. 539576) or 500 U CaMK II (NEB, cat. no. P6060L) or 25 ng CaMK Id (BioVision, cat. no. 7713–5) or 25 ng MST2 (Millipore, cat. no. 14–524) in kinase reaction buffer (30 mM Hepes pH 7.5, 100 mM NaCl, 2 mM MgCl₂, 300 μM ATP, 2 mM DTT [+1mM CaCl₂ and 1 μg/ml (approx. 3 μM) Calmodulin (NEB, cat. no. 6060S) for CaMK II/Id]). Samples were incubated for 3 h at 30°C in a PCR cycler and the reaction was stopped by addition of SDS sample buffer. For visualisation of phosphoprotein, 2 μg of obscurin substrate were separated by SDS-PAGE and stained with Pro-Q^TM Diamond Phosphoprotein Gel Stain as described below. For preparative MST2-phosphorylation of obscurin SH3-DH or DH_5667-5899, 500 ng MST2 were added to a reaction volume of 200 μL kinase reaction buffer containing a substrate concentration of 110 μM and the reaction was allowed to proceed for 5 hrs at 25°C, followed by further incubation overnight at 8°C. The phosphorylated protein was buffer exchanged into 40 mM Hepes pH 7.5, 100 mM NaCl and 2 mM DTT.