Fig. 2: Aurora B phosphorylates RPA at Ser-384 specifically in mitosis.

a Western blot analysis of asynchronous (Async) HCT116 cells, nocodazole-arrested mitotic cells, and arrested cells released (3 h) into G1 phase. Blots were probed with monoclonal phospho Ser-384 RPA70 (pS384-RPA70) (custom antibody, Genscript) and total RPA70 antibodies. Vinculin was used as a loading control. Mitotic arrest was confirmed using phospho-Ser10-histone H3 specific antibody (pS10-HH3). * Indicates a slower migrating form of RPA70 enriched in mitotic cells. b Similar mitosis-specific phosphorylation of RPA70 at Ser-384 was also observed in 293 T cells using western blot analysis. All subsequent studies were carried out in HCT116 cells. c RPA70 phosphorylation at Ser-384 was assessed in different phases of the cell cycle by synchronization at G1/S boundary using double thymidine block followed by release into S and G2 phases. Phosphorylation in mitosis was assessed as described in a). Tubulin and vinculin were used as loading controls. Specific phosphorylation of RPA70 at Ser-384 is observed only during mitosis. d Aurora B was selectively inhibited in mitotic cells with short-term treatment (45 min) of 3 μM AZD1152 and results in loss of RPA70 Ser-384 phosphorylation. Inhibition of Aurora B was confirmed by loss of Ser10-Histone H3 phosphorylation. Asynchronous cells and vehicle-treated (0.1% DMSO) mitotic cells were used as controls. e Cells were treated with vehicle (Veh; 0.1% DMSO) or 10 ng/ml SN-38 for 21 h to induce replication stress. DNA damage does not induce RPA70 phosphorylation at Ser-384. All blots in this figure are representative of at least three independent replicates.