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. 2015 Jan 31;6(14):11779–11793. doi: 10.18632/oncotarget.3151

Figure 4. COP1 modification is required for DNA damage-mediated p27 accumulation.

Figure 4

(A) p27 accumulates in A549 cells after DNA damage. A549 cells were treated with 10 μg/ml irinotecan and 20 μg/ml cyclophosphamide for the indicated times and lysates were analyzed by IB with the indicated antibodies. (B) p27 accumulation occurs regardless of construct modifications. A549 cells were transfected as indicated with wild-type (wt) Flag-p27 or T157A, T187A, ΔJab1, or ΔJab1+T187A mutants. Cells were treated with 1 μg/ml DOX for the indicated times. Equal amounts of protein from cell lysates were analyzed by IB with anti-Flag, anti-COP1, or anti-Actin. (C) Skp2 is not involved in DNA damage-mediated p27 accumulation. Wild-type (wt) MEF cells and Skp2−/− cells were treated with 1 μg ml doxorubicin (DOX) for the indicated times. Lysates were immunoblotted with the indicated antibodies. (D) COP1 S387A mutant binds to p27 with less efficiency. Cells were transfected with the indicated plasmids and equal amounts of lysates were immunoprecipitated with anti-GFP, followed by immunoblotting with indicated antibodies. (E) COP1 S387A mutant reduces the steady-state expression of p27 with less efficiency. Cells were transfected with the indicated plasmids and equal amounts of lysates were immunoblotted with anti-Flag to examine the expression of p27. (F) COP1 S387A mutant accelerates the turnover of p27 with less efficiency. Cells were transfected with the indicated plasmids and treated with CHX (100 μg/ml) for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. (G) COP1 S387A mutant does not have inverse relationship with p27 in the presence of DNA damage. PC3 cells were transfected with the indicated plasmids and cells were treated with 1 μg/ml doxorubicin (DOX) for the indicated times. Lysates were immunoblotted with the indicated antibodies. (H) COP1 S387A mutant does not have impact on p27 ubiquitination in the presence of DNA damage. PC3 cells were transfected with the indicated plasmids and cells were treated with 1 μg/ml doxorubicin (DOX) for the indicated times as in (g). The cell lysates of the transfected cells from (g) were immunoprecipitated with anti-ubi and immunoblotted with an anti-p27 antibody.