Fig. 2: β-TrCP binds to TOP2β and negatively regulates TOP2β levels.

a Evolutionary conservation of the β-TrCP degron motifs of TOP2β. b β-TrCP silencing abrogates TOP2β reduction by VM-26 treatment. Cells transfected with siRNA oligos targeting both β-TrCP1 and β-TrCP2 (siβ-TrCP1 + 2) or scrambled control siRNA (siCtrl) were left untreated or treated with VM-26 (100 μM) for 2 h, and then, IB was undertaken with the indicated Abs. c β-TrCP silencing suppresses the TOP2β degradation by VM-26 treatment in a time-dependent manner but has no effect on TOP2α. Cells transfected with siRNA oligos targeting β-TrCP or scrambled control siRNA were treated with VM-26 for the indicated time periods, and then, IB was undertaken with the indicated Abs. d β-TrCP silencing extends TOP2β protein half-life upon VM-26 treatment. Cells transfected with the indicated siRNA were treated with CHX and VM-26 for the indicated time periods and then subjected to IB with the indicated Abs. Densitometry quantification was performed with ImageJ, and the decay curves are shown (bottom). e Ectopically expressed β-TrCP1 binds to endogenous TOP2β. HEK293 cells transfected with the indicated plasmids were left untreated or treated with VM-26 and MG132 (20 μM) for 5 h. Cells were then harvested for IP with anti-FLAG beads (top) and direct IB with the indicated Abs (bottom). f β-TrCP1 binds to endogenous TOP2β. HeLa cells were lysed and subjected to IP with β-TrCP1 antibody or normal rabbit immunoglobulin (IgG), and then, IB was undertaken with anti-TOP2β Ab (top). Cell lysate was also directly immunoblotted with the indicated Abs (bottom). g β-TrCP1 promotes TOP2β polyubiquitination in vivo. HEK293 cells transfected with the indicated plasmids were treated with MG132 and VM-26 for 5 h, and then, IP was conducted with anti-HA beads (top), and direct IB was undertaken with the indicated Abs (bottom).