Figure 6.

Chk1 is phosphorylated on S345 after DNA damage and this is regulated by Atr. (A) Phosphorylation of Chk1 on S345 after DNA damage. 293T cells were untreated or treated with IR (20 Gy and harvested after 1 hr), UV (50 J/m² and harvested after 2 hr) or HU (1 mm for 24 hr). Whole cell lysates were immunoprecipitated (IP) with rabbit anti-Chk1 or anti-p-S345 antibodies followed by immunoblotting with mouse anti-Chk1 antibodies. (B) Overexpression of wild-type (WT) but not kinase-deficient (KD) Atr enhances S345 phosphorylation of cotransfected Chk1. Thirty-six hours after cotransfection of CMV:Chk1 and CMV:FLAG-Atr–WT or CMV–FLAG–Atr–KD, 293T cells were untreated (−) or treated with UV (50 J/m²) and harvested after 1.5 hr. Whole cell lysates were immunoblotted with anti-FLAG antibodies to detect Atr expression, or assayed for S345 phosphorylation of Chk1 by IP–Western blot as described in (A). (C) Induction of kinase-deficient Atr inhibits S345 phosphorylation of endogenous Chk1. GM847/ATR–KD cells were cultured in the absence (−) or presence (+) of 1 μg/ml doxycycline (Dox) for 48 hr, untreated (−) or treated with UV (50 J/m²) and harvested after 1 (1h) or 4 (4h) hr. Whole cell lysates were immunoblotted with anti-FLAG or mouse anti-Chk1 antibodies to detect Atr–KD or endogenous Chk1 expression. And IP–Western was performed to detect S345 phosphorylation of Chk1 as described in A.