In vitro kinase assays were performed using 20 μl of affinity-purified Flag-BRK-YF and a 10-μl volume of affinity-purified substrate (SMAD4) in a reaction volume of 50 μl, comprising 20 μl of kinase buffer [25 mM tris-HCl (pH 7.5), 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2; kinase buffer (10×) #9802, Cell Signaling Technology] with or without 200 μM ATP. The reaction was carried out at 30°C for 30 min and terminated by the addition of Laemmli sample buffer. The samples were then boiled for 10 min and resolved via SDS-PAGE.

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