Skp2-dependent ubiquitination of Nbs1 occurs in cells treated with NCS.A, stability of Nbs1 and Mre11 proteins in RecQL4- or mock-depleted U2OS cells treated with NCS for indicated times. All cells were treated with 50 μg/ml cycloheximide. The levels of Mre11 and Nbs1 proteins normalized to that of Lamin B1 (LB) from three independent experiments are shown below each lane. B, ubiquitination of Nbs1 in 293T cells depleted of RecQL4 and/or Skp2, 1 h after NCS or mock treatment. Nbs1 proteins were immunoprecipitated with anti-FLAG antibody, and anti-HA (for ubiquitin), anti-K48 ubiquitin (K48), and anti-FLAG (for Nbs1) antibodies were used for Western blotting. C, Western blots showing depletion of Skp2 and RecQL4 in cells used in (B). NCS, neocarzinostatin.