FIG. 3.

Checkpoint-induced phosphorylation of Chk1 is ATM independent but caffeine sensitive. (A) AT⁺ and AT⁻ cells were untreated (lanes 1 and 4) or were treated with 3 mM HU (lanes 2 and 5) or irradiated with 10 Gy of IR (lanes 3 and 6). Cellular lysates containing 150 μg of protein were resolved directly by SDS-PAGE on an 8% polyacrylamide gel. Chk1 was detected by Western blotting with Chk1-specific polyclonal antibody (SC7898). (B) HeLa cells were cultured in DMEM lacking caffeine (lanes 1 and 2) or containing 10 mM caffeine (lanes 3 and 4) for 2.5 h. Cells were cultured for an additional 20 h in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 3 mM HU. Cellular lysates containing 150 μg of protein were resolved directly by SDS-PAGE on an 8% polyacrylamide gel. Chk1 was detected by Western blotting with Chk1-specific polyclonal antibody (SC7898).