Fig 2: Mtp53 destabilizes DET1 and blocks the interaction between DET1 and ETS2(A, B, top) A549 cells were transfected with the indicated plasmids and protein turnover was assessed after blocking new protein synthesis with CHX. (A, bottom) Densitometry analysis for relative DET1 or COP1 (B, bottom) protein levels normalized to the 0 h timepoint for each tranfection. (C) Co-immunoprecipitation of ETS2 with COP1, DET1, with or without mtp53. PC3 cells were transfected with ETS2, COP1, DET1, and mtp53 R248W for 24 h. Samples were treated with MG-132 for 6 h prior to harvesting. Lysates were then immunoprecipitated with anti-ETS2 (C-20) antibodies and then processed for Western blotting. Solid triangle indicates increasing amounts of plasmid.

Fig 3: Alanine mutations of CDK10 serine phosphorylation sites in ETS2 disrupt DET1 binding and reduces COP1 interactionCo-immunoprecipitation of ETS2 wild-type or the mentioned alanine mutants with DET1 (A) and COP1 (B). PC3 cells were transfected with either wild-type ETS2 or the mentioned alanine mutants and DET1 (A) or COP1 (B) for 24 h. Samples were treated with MG-132 for 6 h prior to harvesting. Lysates were then immunoprecipitated with anti-ETS2 (C-20) antibodies (A) or anti-COP1 antibodies (B) and then processed for Western blotting.

Fig 4: COP1/DET1 aid in the CDK10 mediated degradation of ETS2Degradation of ETS2 wild-type and alanine mutants by COP1/DET1 and CDK10. (A) PC3 cells were transfected with wild-type ETS2, CDK10, and a set amount of CDK10 with increasing amounts of COP1 for 24 h. (B) PC3 cells were transfected withwild-type ETS2, CDK10, and a set amount of COP1 with increasing amounts of CDK10. (C) A549 cells were transfected with either wild-type ETS2 or with the mentioned ETS2 alanine mutants and CDK10 for 24 h. (D) A549 cells were transfected with either wild-type ETS2 or with the mentioned ETS2 alanine mutants, COP1 and DET1, for 24 h. (A, B, C, D) Lysates were then harvested and processed for Western blotting. Solid triangle indicates increasing amounts of plasmid.

Fig 5: ETS2 amino acids V52, P53 and V308, P309 correspond to COP1 binding motifs required for degradation and interactionDegradation of ETS2 deletion and VP mutants by COP1/DET1. (A) A549 cells were transfected with either full-length ETS2 or with the mentioned ETS2 deletion constructs, COP1 and DET1, for 24 h. (B, C) A549 cells were transfected with either wild-type ETS2 or with the mentioned ETS2 VP constructs, COP1 and DET1 for 24 h. Lysates were then harvested and processed for Western blotting. (D) Lysates of PC3 cells transiently expressing COP1, DET1 and the mentioned ETS2 VP mutants were immunoprecipitated with anti-ETS2 (C-20) antibodies. The immunoprecipitates were processed for Western blotting with anti-Myc antibodies.