Protein kinase buffer

SYK (Sigma-Aldrich, #S6448), cortactin (Abcam, Cambridge, United Kingdom, #ab131824), and cofilin (Abcam, #ab95396) recombinant proteins were phosphorylated in protein kinase buffer (New England Biolabs, Ipswich, MA, USA) with 100 μM ATP. The reactions were incubated at 37°C for 1 h, and the products were separated on SDS-PAGE gels. To estimate the amount of ADP produced in the kinase reactions, ADP-Glo Kinase assay (Promega, Madison, WI, USA) was used according to the manufacturer’s instructions. Luminescence generated by reactions performed in the absence of SYK were subtracted to eliminate any signal from background auto-phosphorylation of proteins.

Condensin I was purified from 293T cells expressing p3×Flag-CMV10-NCAP-H as described above and dephosphorylated with 200 U of λ-phosphatase (New England Biolabs). λ-phosphatase was inhibited by the addition of 5 mM sodium vanadate, and condensin I was incubated with 125 U of CK2 (New England Biolabs) in protein kinase buffer (New England Biolabs) supplemented with 200 mM ATP (Sigma) at 30°C for 1 hour. Reactions were quenched by the addition of SDS-PAGE sample buffer, reduced, and alkylated (as described above), and then separated by SDS-PAGE gel electrophoresis. Bands corresponding to NCAP-G were excised and analyzed by proteinase K digest and reductive dimethyl labeling as described above, as well as by LC-MS/MS.