Skip to main content
Fig. 5 | BMC Biology

Fig. 5

From: The CRK2-CYC13 complex functions as an S-phase cyclin-dependent kinase to promote DNA replication in Trypanosoma brucei

Fig. 5

CRK2 phosphorylation of Thr-310 in Mcm3 is essential for cell proliferation. a Schematic flowchart illustrating the Mcm3-3′UTR RNAi complementation strategy. The 3′UTR* in the red box indicates RNAi-resistant 3′UTR sequence from a different gene. b Analysis of the sequences flanking the codon encoding wild-type and mutated Thr-310 residue in the replaced Mcm3 locus. c Western blotting to monitor the knockdown of Mcm3, which was tagged with a PTP epitope, and the expression of RNAi-resistant wild-type and mutant Mcm3 proteins, which was tagged with a triple HA epitope. in non-induced control and Mcm3-3′UTR RNAi-induced cells. PTP-Mcm3 was detected by anti-protein A antibody, whereas Mcm3-3HA, Mcm3T310A-3HA, and Mcm3T310D-3HA were detected by anti-HA antibody. TbPSA6 served as a loading control. d Effect of CRK2-mediated phosphorylation of Thr-310 in Mcm3 on cell proliferation. Shown are growth curves of Mcm3-3′UTR RNAi cell line and Mcm3-3′UTR RNAi complementation cell lines. e Effect of Mcm3 knockdown and Mcm3 RNAi complementation by wild-type and mutant Mcm3 on EdU incorporation. Shown is the quantitation of EdU-positive 1N1K cells before and after tetracycline induction for 48 h. Error bars indicate S.D. from three independent experiments (n = 3). ***p < 0.001; ns, no significance (chi-square test)

Back to article page