Ser220 and Ser251 are the critical sites for M phase specific modifications of SOX2. PA-1 cells expressing HA tagged wild type or mutants SOX2 (HA-SOX2) were incubated with DMSO or nocodazole for 12 hours to arrest cells at mitosis. VX680 were added (for 30 minutes) for AURKA inhibition. AURKA inhibition can inhibit the mitotic phosphorylation of SOX2. Consistently, mutations on Ser220 and Ser251 can also completely abolish the mitotic phosphorylation of SOX2. For other mutants, including S37A, T116A, S228A, S246A, S249A, and S250A, they did not show any effect on the mitotic phosphorylation of SOX2.