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. 2012 Mar 12;109(13):4828-4833. doi: 10.1073/pnas.1116349109

Fig. 3.

Fig. 3.

UHRF1 S652 is phophorylated in the M phase and is likely mediated by CDK1-cyclin B. (A) FLAG-UHRF1 was purified from transiently transfected 293T cells, digested with trypsin, and analyzed by LC. MS/MS spectrum of the peptides at m/z 836.83, identifying EEEEQQEGGFASPR phosphorylation on S652. (B) 293T cells were synchronized to G1/S by double thymidine block and then released. Cells collected at indicate time points were lysated for Western blot analysis using UHRF1, pS652, CCNB1, and actin antibodies. (C) HCT116 p53-/- cells were fixed and immunostained with the UHRF1 and S652 phospho-UHRF1 antibodies while DNA was stained with DAPI. In the immunostaining image some of the cells that are undergoing mitosis and show clear UHRF1 S652ph signals are circled. The same cells in the DAPI-stained image are indicated by arrows. Out of a total of 529 S652ph-positive cells counted, 428 showed clear chromosome condensation (91%) indicating that they are in the M phase of the cell cycle. Scale bars, 10 μm. (D) Wild-type UHRF1 (aa 600–687) and S652A mutants were purified from bacteria and subjected to phosphorylation by CDK1-cyclin B (purchased from NEB) in vitro and were analyzed by Western blotting using the S652 phospho-UHRF1 antibody. (E) HCT116 p53-/- cells were treated with 50 μM roscovitine (ROS) for the indicated time. Lysates were analyzed by Western blot using the S652 phospho-UHRF1, UHRF1, and actin antibodies. The S652 phospho-UHRF1 and UHRF1 band intensity was normalized against the internal actin controls.