FIGURE 4.

Lysine-free Otub1 attenuates p53 activation in response to DNA damage. A and B, Otub1^K0, but not Otub1^wt, attenuates p53 activation following Eto treatment. U2OS-TO-FLAG-Otub1^K0 cells (A) and U2OS-TO-FLAG-Otub1^wt cells (B) were cultured in the absence or presence of Dox for 24 h, followed by treatment with Eto (20 μm) or control dimethyl sulfoxide for the indicated times, followed by IB analysis. C, reintroduction of Otub1^wt, but not the Otub1^K0 mutant, rescues the p53 response following DNA damage in cells with Otub1 knockdown. U2OS cells transfected with control, the FLAG-Otub1^wt or FLAG-Otub1^K0 plasmid, and scrambled or Otub1 siRNA targeting the 3′ UTR of the Otub1 mRNA are shown, as indicated. The cells were treated with Eto (20 μm) for 6 h before harvesting 48 h after siRNA transfection, followed by IB analysis. D and E, Otub1^K0 attenuates p53 activation in response to DNA damage. U2OS-TO-FLAG-Otub1^K0 cells were cultured in the absence or presence of Dox for 24 h, followed by treatment with Eto (20 μm) or control (Ctl) dimethyl sulfoxide for 6 h, and assayed by RT-qPCR (D) and cell cycle profile analysis for sub-G₁ phase cells (E).