S33-RPA2 phosphorylated by ATR in response to replication stress. (A,B) The
M059J (DNA-PK–/–) or M059K
(DNA-PK+/+) paired cell lines (A), and GM637
(ATM+/+) or GM5849
(ATM–/–) lines (B) were either mock-treated or
treated with 2.5 mM HU for 3 hours to cause replication stress. (C) U2-OS
cells were transfected with an siRNA directed against ATR or a
control siRNA against luciferase (Luc). After 48 hours, cells were either
mock-treated or treated with 2.5 mM HU for 3 hours. (D) U2-OS cells were
transfected with an siRNA directed against Rad17 or luciferase. For all
panels, cells were lysed following treatment and the lysates subjected to
SDS-PAGE and western blotting using antibodies specific to total RPA2,
S33-P-RPA2, ATR, Rad17 or S317-P-Chk1, as indicated. The
total RPA2 signal was used as a loading control. The positions of the various
RPA2 species are indicated as in the Fig.
1 legend.