Fig. 6

Phosphorylation of Thr-310 in Mcm3 by CRK2 promotes its interaction with Sld5. a Effect of CRK2 RNAi on Mcm2-Mcm3 interaction. Shown is the co-immunoprecipitation of Mcm2-PTP and Mcm3-3HA from control and CRK2 RNAi cells. Mcm2-PTP alone and Mcm3–3HA alone were included as negative controls. b Effect of CRK2 RNAi on Cdc45-Mcm3 interaction. Shown is the co-immunoprecipitation of Cdc45-PTP and Mcm3-3HA from control and CRK2 RNAi cells. Cdc45-PTP alone and Mcm3–3HA alone were included as negative controls. c Effect of CRK2 RNAi on Sld5-Mcm3 interaction. Shown is the co-immunoprecipitation of PTP-Sld5 and Mcm3-3HA from control and CRK2 RNAi cells. PTP-Sld5 alone and Mcm3-3HA alone were included as negative controls. The histogram below the western blots is the quantitation of Mcm3 band intensity, which was normalized against that of Sld5 from three independent experiments (n = 3). Error bars indicated S.D. ***p < 0.001 (chi-square test). The faster migrating band in the blot of PTP-Sld5 is a degradation product of PTP-Sld5. d Effect of CYC13 RNAi on Sld5-Mcm3 interaction. Shown is the co-immunoprecipitation of PTP-Sld5 and Mcm3-3HA from control and CYC13 RNAi cells. PTP-Sld5 alone and Mcm3-3HA alone were included as negative controls. The histogram below the western blots is the quantitation of Mcm3 band intensity, which was normalized against that of Sld5 from three independent experiments (n = 3). Error bars indicated S.D. *p < 0.05 (chi-square test). e Effect of Mcm3 Thr-310 phosphorylation on its interaction with Sld5. Shown is the co-immunoprecipitation of PTP-tagged Sld5 and 3HA-tagged wild-type and mutant Mcm3 proteins from T. brucei. The histogram below the western blots is the quantitation of the band intensity of wild-type and mutant Mcm3, which was normalized against that of Sld5 from three independent experiments (n = 3). Error bars indicated S.D. **p < 0.01; ***p < 0.001 (chi-square test)