Fluochem fc2

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).

After treatment, the cells were washed with ice-cold 1 × PBS and lysed with cell lysis buffer (pH 7.4). The cell lysates were incubated on ice for 30 min and the homogenates were centrifuged at 13,000 × g for 10 min at 4℃. The supernatants were collected and protein concentration was determined using the Bradford protein assay. Protein samples (50 µg) were mixed with 2 × loading buffer, and boiled at 100℃ for 5 min. The samples were separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and electrotransferred to nitrocellulose membranes (WhatmanTM, GE Healthcare, Little Chalfont, UK). The membranes were blocked with 5% nonfat dry milk in 1 × PBST buffer (0.1% Tween 20 in PBS) for 1 hr at room temperature and incubated overnight with primary antibodies in blocking buffer at 4℃. The membranes were washed, followed by incubation with anti-rabbit-IgG or anti-goat-IgG with horseradish peroxidase for 1 hr at room temperature. After washing the membrane, final detection and quantification were performed using a chemiluminescence detection system (FluoChem® FC2, Alpha Innotech, CA, USA).