Briefly, the phosphorylation reactions contained 20 mM tris (pH 7.5), 20 mM Hepes (pH 7.5), 5 mM β-glycerophosphate, 1 mM dithiothreitol, 2 mM Na3VO4, 100 mM NaCl, 10 mM MgCl2, 0.1 mM adenosine triphosphate (ATP), LATS2 kinase (SignalChem, L02-11G), 100 ng of recombinant GST and GST-CTCF (WT or mutant) purified from bacteria, and were incubated at 30°C for 1 hour. The reaction mixture was separated by SDS-PAGE, and phosphorylated proteins were detected by Western blotting with anti-RXXpS/T antibody.

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