Differential Dynamics of ATR-Mediated Checkpoint Regulators

Figure 5

Mobility of GFP-Chk1 in response to DNA damage. (a) U2OS cells expressing GFP, H2B-GFP-Chk1, or GFP-Chk1 were left untreated or treated with UV and analyzed by strip-FRAP (see Section 2 for technical details). (b) Chromatin fractionation of GFP-Chk1 expressing cells transfected with siRNA oligos directed against luciferase or Chk1 for 24 hours before UV treatment and cell fractionation. Relative Chk1 levels as compared to untreated controls are indicated. Western blot analysis of the fractions was performed using the indicated antibodies. (c) GFP-Chk1 expressing cells were transfected with siRNA oligos against luciferase or Chk1 for 24 hours and left untreated or treated with UV. One hour later, cells were analyzed by strip-FRAP. (d) U2OS cells expressing GFP-Chk1 were transfected with Chk1 siRNA oligos for 24 hours and left untreated or treated with UV and subsequently analyzed by FLIP-FRAP (see Section 2 for technical details).