Figure 1.

Experimental design for identification of CK2 targets in mitotically arrested HeLa cells. (A) Time course and dosage titration of CX-4945 in mitotically arrested HeLa cells. Cells were treated at 5 μM for times shown or for 45 min with concentrations indicated, collected, and analyzed by western blot using α-phospho-CK2 substrate (pS/pT-D-X-E) or α-actin antibodies. (B) Phosphoproteomic screen scheme for identification of mitotic substrates. Briefly, cells were treated with vehicle control or CX-4945, mixed, lysed, reduced, alkylated, and digested with trypsin. Peptides were then phospho-enriched using titanium dioxide microspheres, separated using pentafluorophenyl reverse-phase fractionation and analyzed by LC-MS/MS.