Figure 2. The interaction between PCNA... | Rockefeller University Press

$Figure 2. The interaction between PCNA and SET8 is important for CDT2-mediated degradation of SET8. (A) Schematic presentation of SET8. Two PIP boxes (PCNA-interacting domains) have been identified N terminally to the SET domain. (B) Mutation of PIP2 results in stabilization of SET8. HEK293 cells were transfected with plasmids expressing Flag-HA-SET8, Flag-HA-SET8-*PIP1, and Flag-HA-SET8-*PIP2. Cells were treated with cycloheximide and processed for immunoblotting with the indicated antibodies. MCM7 was used as a loading control. (C) Increased levels of PCNA result in degradation of WT SET8 but not SET8-*PIP2. HEK293 cells were transfected as illustrated and processed for immunoblotting with the indicated antibodies. (D) In vivo ubiquitylation of SET8-*PIP2 is reduced compared with SET8 WT. HEK293 cells were transfected with Flag-HA-SET8, Flag-HA-SET8-*PIP2, and HIS-ubiquitin as indicated. Cells were processed for immunoprecipitation using cobalt beads and immunoblotted with the indicated antibodies. (E) SET8 is ubiquitylated on chromatin. U2-OS cells were either treated with MG132 for 3 h or left untreated. Soluble and chromatin-bound fractions were analyzed by immunoblotting with SET8 antibody. *, nonspecific band. (F) SET8 WT and PCNA colocalize on chromatin after MG132 treatment. U2-OS cells expressing Flag-HA-SET8 or Flag-HA-SET8-*PIP2 were seeded on coverslips and treated with MG132 for 3 h before preextraction and fixation. Cells were stained with antibodies against HA and PCNA. Bars, 10 µm.$

Figure 2.

The interaction between PCNA and SET8 is important for CDT2-mediated degradation of SET8. (A) Schematic presentation of SET8. Two PIP boxes (PCNA-interacting domains) have been identified N terminally to the SET domain. (B) Mutation of PIP2 results in stabilization of SET8. HEK293 cells were transfected with plasmids expressing Flag-HA-SET8, Flag-HA-SET8-*PIP1, and Flag-HA-SET8-*PIP2. Cells were treated with cycloheximide and processed for immunoblotting with the indicated antibodies. MCM7 was used as a loading control. (C) Increased levels of PCNA result in degradation of WT SET8 but not SET8-*PIP2. HEK293 cells were transfected as illustrated and processed for immunoblotting with the indicated antibodies. (D) In vivo ubiquitylation of SET8-*PIP2 is reduced compared with SET8 WT. HEK293 cells were transfected with Flag-HA-SET8, Flag-HA-SET8-*PIP2, and HIS-ubiquitin as indicated. Cells were processed for immunoprecipitation using cobalt beads and immunoblotted with the indicated antibodies. (E) SET8 is ubiquitylated on chromatin. U2-OS cells were either treated with MG132 for 3 h or left untreated. Soluble and chromatin-bound fractions were analyzed by immunoblotting with SET8 antibody. *, nonspecific band. (F) SET8 WT and PCNA colocalize on chromatin after MG132 treatment. U2-OS cells expressing Flag-HA-SET8 or Flag-HA-SET8-*PIP2 were seeded on coverslips and treated with MG132 for 3 h before preextraction and fixation. Cells were stained with antibodies against HA and PCNA. Bars, 10 µm.

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