Skip to main content
. 2016 Jan 15;15(1):95–105. doi: 10.1080/15384101.2015.1121323

Figure 2.

Figure 2.

MMSET is degraded by CRL4Cdt2. (A) HEK293T cells were transfected with the plasmids denoted above each lane. Lysates were prepared 48 hours post transfection, immunoprecipitated with anti-Flag, and examined for MMSET. Arrowheads denote Flag-YFP (lower) and Flag-Cdt2 (upper). (B) HEK293T cells were transfected with the plasmids denoted above each lane and synchronized in S phase by the serum starvation and release method. Lysates were prepared and examined for the indicated proteins. The numbers beneath the blots provide the densitometric ratio of MMSET signal to Actin, setting the value for the control group as 1. (C) HEK293T cells were transfected as in B, fixed, and stained for either Cdt2 (Flag, red) or MMSET (green). Nuclei were visualized with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, blue). Scale bar= 10 μm. (D) HEK293T cells were treated with the indicated siRNAs for 24 hours, followed by an aphidicolin block to synchronize cells into S phase. Cells were released into S phase for 4 hours in the presence of cycloheximide and lysates were prepared and examined for the indicated proteins. The numbers beneath the blots provide densitometric ratio of MMSET or Cdt2 signal to Tubulin, setting the value for the control group as 1. *Denotes a non-specific band detected by the Cdt2 antibody. (E) His-pull-downs 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. (F) His-pull-downs 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.