FIGURE 4.

Ser-205 is the major site of MCM3 phosphorylated by Chk1. A, HA-MCM3 WT proteins were collected by IP from HEK293T cells, treated with CIAP, used in the in vitro kinase reaction, and run on SDS-PAGE, and the corresponding band was excised and used for mass spectrometry analyses. Phosphorylation was detected for the peptide 203–222 (one of the following four residues: Tyr-204, Ser-205, Thr-208, and Thr-209). B, HEK293T cells were transfected with HA-MCM3 FL (WT, S205A, or ASDLAA) for 48 h, IPed with anti-HA antibodies, and blotted with the anti-Ser(P)/Thr(P) mixture. The same membrane was stripped and reblotted with anti-HA antibodies.