Fig. 4

Mcm3 is a substrate of CRK2. a Yeast two-hybrid assays to test the potential interaction between CRK2 and the subunits of the CMG complex. 2DO, double drop-out; 3DO, triple drop-out. b GST pull-down to test the interaction between CRK2 and the CMG subunits Mcm3 and Sld5. GST-Mcm3 and GST-Sld5 were expressed and purified from bacteria, and CRK2-3HA was expressed in T. brucei. GST alone was used as the negative control. CBB, coomassie brilliant blue. c In vitro kinase assay of Mcm3 phosphorylation by CRK2 in the presence and absence of CYC13. GST-Mcm3, GST-CRK2, and GST-CYC13 were expressed and purified from E. coli. Arrows indicate phosphorylated GST-Mcm3 (GST-pMcm3), which was detected by the anti-ThioP antibody. GST-fused Mcm3, CRK2, and CYC13 were stained with coomassie brilliant blue. The asterisks indicate a non-specific band detected by the anti-ThioP antibody in the reactions to which purified GST-CYC13 was added. d MS/MS spectrum of Ser-213 (upper panel) and Thr-310 (lower panel) phosphopeptides identified from CRK2-phosphorylated GST-Mcm3. In vitro phosphorylated recombinant GST-Mcm3 was analyzed by LC-MS/MS. The phosphorylated Ser-213 and Thr-310 residues are shown in red