Figure 1.

MMSET is regulated in a cell-cycle-dependent manner and degraded by the ubiquitin-proteasome pathway during S phase. (A) Asynchronous U2OS cells or cells synchronized at the G1-S border were released into S phase for the indicated times. Lysates were collected for each time point and blotted for the indicated proteins. (B) Asynchronous U2OS cells were plated on coverslips, fixed, and stained for either Cyclin E1 (red) or MMSET (green). Nuclei were visualized with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, blue). Scale bar=10 μm. (C) U2OS cells were synchronized at the G1-S border by double-thymidine block and released into S phase for the indicated times either in the absence (top) or presence (bottom) of MG132. Lysates were collected at each time point and blotted for the indicated proteins. (D) His-pulldowns were performed under denaturing conditions using lysates from HEK293 cells transfected with the indicated plasmids and synchronized in S phase using the serum starvation and release method. Purified species were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. Arrowhead and a bar indicate monoubiquitinated and polyubiquitinated MMSET, respectively. Data shown are representative of 3 independent experiments.