P cdc25c

EESR-treated cells were lysed with lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol bis(2-aminoethyl ether) tetraacetic acid, 1% Triton X-100, 1 μg/mL leupeptin, 1 mM phenylmethanesulfonyl fluoride) for 1 hour at 4°C and centrifuged for 30 minutes at 13,000 rpm. Total soluble proteins in the supernatant were collected and the concentration of protein was determined by Bradford method. For Western blot analysis, 30 to 50 μg/mL of proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. Blots were incubated at 4°C overnight with specific primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies and visualized by an enhanced chemiluminescence detection system (FluoChem®FC2; Alpha-Innotech, San Leandro, CA, USA) using Western blotting luminol reagent (Santa Cruz Biotechnology, Dallas, TX, USA). CDK1, cyclin A, cyclin B, cell division cycle 25C (Cdc25C), p-Cdc25C, Wee1, p53, Fas, Fas-associated protein with death domain (FADD), Bax, caspase-3, caspase-8, caspase-9, PARP, actin primary antibodies and peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology. Primary antibodies against CHK2, p-CHK2, p21, p-CDK1, and Bcl-2 were purchased from Cell Signaling Technology (Beverly, CA, USA).