Figure 2.

Requirement of Rad17 for loading Rad9 onto chromatin and phosphorylation of Chk1. (A) 293T cells were cotransfected with plasmids expressing GFP–Rad9 and either vectors alone or plasmids expressing Flag–Rad17. After 48 h, cells were untreated or treated with 50 J/m² of UV and harvested after 2 h. Fractions from these cells were analyzed by immunoblotting with the indicated antibodies. (S1+S2) A combined fraction of S1 and S2. (B) HeLa cells were either transfected with siRNA targeting Rad17 or mock-transfected, and untreated or treated with UV as in A. Fractions from these cells were analyzed by immunoblotting. (C) HeLa cells transfected with siRNA targeting Rad17 or mock-transfected HeLa cells were untreated or treated with 50 J/m² of UV and harvested after 2 h. Extracts of the cells were immnoprecipitated with an anti-p-S345 Chk1 antibody (Liu et al. 2000). Both extracts (WCE) and immunoprecipitates (lower panel) were analyzed by immunoblotting.