FIG. 5.
Chk2 oligomerization. (A) 293 cells stably expressing Flag-Chk2 were transiently transfected with HA-Chk2. Transfectants were treated with 1 mM hydroxyurea for 24 h, beginning 24 h after transfection, or exposed to gamma irradiation (20 Gy) or UV (50 J/m2) 48 h after transfection. Cell lysates were harvested 24 h after hydroxyurea treatment or 2 h after irradiation. Upper panel, expression and mobility shift of HA-Chk2 were determined by immunoblotting lysates with anti-HA antibody. Lower panel, coimmunoprecipitation with anti-Flag antibody was performed with equal portions of cell lysates and detected with anti-HA. (B) ATM-dependent increase in Chk2 coimmunoprecipitation. GM5849C ataxia-telangiectasia cells were transiently transfected with HA-Chk2 and Flag-Chk2, plus vector (pcDNA3), wild-type Flag-ATM, or kinase-defective Flag-ATMkd. Transfectants were exposed to 20 Gy of gamma irradiation 48 h after transfection. Cell lysates were harvested 2 h after irradiation. Equal amounts of lysates were immunoprecipitated with anti-HA (top and bottom panels) or anti-Flag (middle panel) antibodies. Precipitates were blotted for Flag-Chk2 (top two panels) or HA-Chk2 (bottom panel). IP, immunoprecipitation; IB, immunoblotting; Ab, antibody.