Figure S7. Cdt2 PIP box is important for CRL4^Cdt2 activity in U2OS cells.

(A) U2OS stable cells expressing Cdt2^WT or Cdt2^PIP-3A. U2OS cells were transfected with expression vectors for Cdt2^WT-FLAG or Cdt2^PIP-3A-FLAG, and isolated stable clones were verified by immunofluorescent analysis and Western blot analysis. Frequency of expressing cells for each construct was shown (%). (B) Poly-ubiquitination assay with U2OS stable cells. U2OS cells and Cdt2^WT-3FLAG or Cdt2^PIP-3A-3FLAG expressing stable U2OS cells were treated with MG132 for 3 h or not (−), and whole-cell extracts were prepared for Western blotting. (C) Cells were transfected with siCdt2 or control siLuc for 72 h, irradiated with UV (+, 5 J/m²) or not (−), and collected 1h later for Western blotting. Relative levels of Cdt1 were shown, UV (−) level set as 1.0 (average of two independent experiments).