Protease and phosphatase inhibitor

MCF-7 cells were grown in the same manner as described in the previous sections. The medium was removed and the cells were scraped in cold PBS containing proteinase and phosphatase inhibitors (Thermo Scientific) and subsequently pelleted by centrifugation. Each cell pellet was incubated for 10 min in 200 μl of cold buffer A (10 mM Hepes [pH 7.9], 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM DTT, protease and phosphatase inhibitors (Thermo Scientific) supplemented with 0,1% Triton X-100. The samples were subjected to low-speed centrifugation (4 min, 1300 g, 4°C) to collect the nuclei, which were subsequently washed in 200 μl of buffer A. The pelleted nuclei were lysed in 200 μl of buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, protease and phosphatase inhibitors (Thermo Scientific)) and kept on ice 30 min. After three washes in buffer B, the chromatin pellets were resuspended in 200 μl of buffer B and sonicated for 30 s (Bioruptor, Diagenode) to shear DNA. Chromatin was pelleted by centrifugation (5 min, 16 000 g, 4°C), and the pellets were resuspended in buffer B supplemented with 4x loading dye (Life Technologies GmbH).

Glutathione-S-transferase-(GST)-tagged FOXC2 truncation mutants were subcloned into pGEX-6P-1 and expressed in Escherichia coli. Cell lysates were cleared by centrifugation and the GST-FOXC2 fusion proteins were absorbed on glutathione–sepharose-4B beads (Sigma) for 2 h at 4 °C. The beads were washed with lysis buffer and the GST-FOXC2 fusion proteins were eluted with reduced glutathione. The eluates (200 ng) were incubated with 100 ng of recombinant active p38α (Invitrogen, Grand Island, NY, USA; PV3304) in the presence of 60 mM MgCl₂, 60 μM ATP, 50 mM Tris-HCl (pH 7.5), 12 mM dithiothreitol, protease and phosphatase inhibitors (Roche) and 0.7 μCi of [γ-³²P]ATP, at room temperature for 30 min.