P6010

rFMRP was diluted to 11.7 µm in CK2 kinase buffer (NEBuffer for Protein Kinases, #B6022; New England Biolabs, Ipswich, MA) and incubated with 4 mm ATP with or without active CK2 (New England Biolabs, #P6010). Reactions (25-µl) were performed according to the manufacturer’s protocol (New England Biolabs). Human recombinant FMRP S500 was generated as previously described (Evans and Mihailescu, 2010 (link)).

Two hundred nanograms of recombinant CK2 (NEB, P6010) and 300 ng of recombinant RUNX2 (Origene, TP760214) were incubated in kinase buffer (20 mM HEPES, pH 7.5, 20 mM MgCl₂, 1 mM EDTA, 2 mM NaF, 2 mM-glycerophosphate, 1 mM DTT, 10 μM ATP) containing 10 μCi of γ32P-ATP (PerkinElmer) for 15 min at 30 °C. The phosphorylated proteins were visualized by autoradiography.
For phospho-mass spectrometry, 1 μg of recombinant CK2 and RUNX2 were incubated in kinase buffer containing cold ATP for 30 min at 30 °C, subjected to SDS-PAGE, and stained with Coomassie blue. Phosphorylated RUNX2 protein at the band size of 55 kDa was excised and subjected to phospho-mass spectrometry.

Phosphorylation reaction containing FANCD2/FANCI and CK2 (P6010, NEB) were incubated at 30°C for 30 min in 50 mM Tris (pH 7.5), 10 mM MgCl₂, 2 mM ATP and 2 mM DTT. Dephosphorylation reaction containing FANCD2/FANCI and λPP (P0753, NEB) were incubated at 30°C for 30 min in 50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM MnCl₂ and 2 mM DTT.