FIGURE 2.

MCM3 phosphorylation dependent on Chk1. A, the same in vitro kinase was performed as in Fig. 1A using either Chk1 WT or a kinase-dead (KD) mutant as the kinase. B, HEK293T cells were transfected with HA-MCM3. After 48 h, cells were treated with 1 or 2 μm Chk1 inhibitor for 6 h, lysed, IPed with anti-HA antibodies, and immunoblotted with the anti-Ser(P)/Thr(P) mixture. The same membrane was stripped and reblotted with anti-HA antibodies. Protein expression in whole cell extracts (WCE) was assessed. C, HEK293T cells were treated or not with 1 μm Chk1 inhibitor (Chk1i) for 12 h, lysed, IPed with anti-MCM3 antibodies, and immunoblotted with the indicated antibodies. D, cells treated with 1 μm Chk1 inhibitor for 6 and 12 h were analyzed for the cell cycle profile.