Compound-interacting proteins were affinity-purified as described previously (22) with modifications. Unsynchronized, confluent U2OS cells (1.6 × 108 cells) were homogenized with a Dounce homogenizer and 5 ml of lysis buffer 1 [25 mM Mops, 15 mM EGTA, 15 mM MgCl2, 1 mM DTT, and cOmplete Protease Inhibitor Cocktail (Roche) (pH 7.2)]. After sonication, the homogenate was supplemented with NP-40 (final 0.5%) and incubated on ice for 5 min, followed by centrifugation (14,000g) at 4°C for 20 min. The resulting supernatant was split into two, and each portion was incubated with 0 or 50 μM GO289 (final 0.5% DMSO) at 4°C for 30 min with rotation. Then, 120 μl of agarose-conjugated GO457 [50% slurry in bead buffer 1 (50 mM tris, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% NP-40, and cOmplete Protease Inhibitor Cocktail (pH 7.4)] was added to the mixture and incubated at 4°C for 1 hour with rotation. Agarose beads were washed six times with 1 ml of bead buffer 1 and boiled with SDS sample buffer to elute bound proteins. Proteins were partially separated (~2 cm) by SDS–polyacrylamide gel electrophoresis (PAGE). The gel lane for each condition was cut horizontally into six pieces (~0.2 cm3 for each piece) and subjected to protein mass spectrometric analysis. For recombinant CK2, 300 ng of CK2 (P6010, New England Biolabs) was diluted with 300 μl of lysis buffer 1 and pulled down with 25 μl of agarose-conjugated GO457.

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