Expression of DNA damage/cell cycle response proteins in unirradiated and irradiated MCF-7 cells. (a) Lysates were prepared from unirradiated monolayer and mammosphere cells and from cells harvested one hour after exposure to 1 or 10-Gy 60Co γ-radiation and analyzed by immunoblotting. Phospho-Chk1 and phospho-Chk2 indicate phosphorylation of Chk1 and Chk2 at Ser345 and Thr68, respectively. Actin served as a loading control. (b) Phosphorylation of p53 at various amino acids. Phosphorylation at serines 6, 9, 37, 46, and 392 was negative (data not shown).