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For MK2 kinase assays, the purified MK2-mycDDK (1 μg) was incubated at 37 °C for 30 min in 1x kinase buffer [containing Tris-HCl (pH = 7.5; 25 mM), β-Glycerophosphate (5 mM), MgCl2 (10 mM), DTT (2 mM), Na3VO4 (0.1 mM), and ATP (200 μM)] plus wild-type OCT4 or mutant OCT4S111A (1 μg each). The reactions were quenched by the addition of 4× LDS loading buffer and 100 mM DTT, followed by boiling at 95 °C for 10 min. The protein samples were resolved on SDS-PAGE and IB with anti-OCT4 antibody.
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