Figure 1. CRL-CDT2 targets SET8 for... | Rockefeller University Press

$Figure 1. CRL-CDT2 targets SET8 for degradation in S phase. (A) SET8 protein level is low in S phase. U2-OS cells were treated with aphidicolin for 24 h and released for 4 h. The indicated cells were treated with MG132 1 h before harvest. Cells were processed for immunoblotting with the indicated antibodies. Vinculin was used as a loading control. Async, asynchronous control cells. Protein levels of SET8 are quantified and depicted below the figure. (B) SET8 contains a conserved PIP box degron. Cluster alignment of SET8 from different species show a highly conserved stretch of amino acids. When this stretch was aligned with CDT1 and p21, it revealed a conserved PIP box degron. (C) SET8 is mutually exclusive with PCNA and CDT2 on chromatin. U2-OS cells were synchronized with nocodazole, released into fresh medium, and collected at the indicated time points. Chromatin-bound fractions and whole cell extracts (WCE) were analyzed by immunoblotting with the indicated antibodies. (D) DDB1 and CDT2 depletion stabilizes SET8. U2-OS cells were treated with the indicated siRNAs 12 h before aphidicolin treatment. 24 h after aphidicolin treatment, cells were washed and released into fresh medium for 3 h and processed for immunoblotting with the indicated antibodies. Actin was used as a loading control. (E) CDT2 is important for the turnover of SET8 in S phase. U2-OS cells were treated with siRNA 12 h before aphidicolin treatment. 24 h after aphidicolin treatment, cells were washed and released into fresh medium for 3 h. Cells were then treated with cycloheximide (CHX) as indicated and processed for immunoblotting with the indicated antibodies. The blots were quantified using an image reader. Representative data from three similar experiments are shown. Data are presented as mean + SD. (F) SET8 is ubiquitylated in vivo mediated by CDT2. HEK293 cells were depleted for CDT2 using siRNA 12 h before transfection with Flag-HA-SET8 and HIS-ubiquitin (HIS-UBQ) as indicated. Cells were processed for immunoprecipitation using cobalt beads and immunoblotted with the indicated antibodies.$

Figure 1.

CRL-CDT2 targets SET8 for degradation in S phase. (A) SET8 protein level is low in S phase. U2-OS cells were treated with aphidicolin for 24 h and released for 4 h. The indicated cells were treated with MG132 1 h before harvest. Cells were processed for immunoblotting with the indicated antibodies. Vinculin was used as a loading control. Async, asynchronous control cells. Protein levels of SET8 are quantified and depicted below the figure. (B) SET8 contains a conserved PIP box degron. Cluster alignment of SET8 from different species show a highly conserved stretch of amino acids. When this stretch was aligned with CDT1 and p21, it revealed a conserved PIP box degron. (C) SET8 is mutually exclusive with PCNA and CDT2 on chromatin. U2-OS cells were synchronized with nocodazole, released into fresh medium, and collected at the indicated time points. Chromatin-bound fractions and whole cell extracts (WCE) were analyzed by immunoblotting with the indicated antibodies. (D) DDB1 and CDT2 depletion stabilizes SET8. U2-OS cells were treated with the indicated siRNAs 12 h before aphidicolin treatment. 24 h after aphidicolin treatment, cells were washed and released into fresh medium for 3 h and processed for immunoblotting with the indicated antibodies. Actin was used as a loading control. (E) CDT2 is important for the turnover of SET8 in S phase. U2-OS cells were treated with siRNA 12 h before aphidicolin treatment. 24 h after aphidicolin treatment, cells were washed and released into fresh medium for 3 h. Cells were then treated with cycloheximide (CHX) as indicated and processed for immunoblotting with the indicated antibodies. The blots were quantified using an image reader. Representative data from three similar experiments are shown. Data are presented as mean + SD. (F) SET8 is ubiquitylated in vivo mediated by CDT2. HEK293 cells were depleted for CDT2 using siRNA 12 h before transfection with Flag-HA-SET8 and HIS-ubiquitin (HIS-UBQ) as indicated. Cells were processed for immunoprecipitation using cobalt beads and immunoblotted with the indicated antibodies.

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