Pcaspase 3

The proteins from cell lysates were separated on 12% SDS–polyacrylamide gels and blotted onto PVDF membranes. All gels have been run under the same experimental conditions. Membranes were then probed with an antibody. Antibodies against sCLU, pCaspase-3, Caspase and Bcl-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against pERK1/2, ERK1/2, pAKT, AKT were purchased from Abcam (Cambridge, MA). We used BCIP/NBT color development system to detect the bands. Images were photographed using the Bio-Rad gel imaging system (Bio-Rad, CA).

For western blot analysis, lysate proteins (45 μg) were separated by 12% or 15% w/v SDS-polyacrylamide gel electrophoresis. The separated proteins were then transferred onto nitrocellulose transfer membranes (0.2 or 0.45 μm, Millipore, Bedford, MA). The membranes were blocked with 5% nonfat dry-milk in a buffer (10 mM Tris-HCL [pH 7.6], 100 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature, incubated with the desired primary antibodies overnight at 4°C and then incubated with alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies at a 1 : 1000 dilution for 1 h at room temperature as previously described [27 (link)]. After washing, the proteins were detected using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Protein levels were quantified by densitometry using Quantity One software (Bio-Rad). Antibodies against β-actin, ERβ, Bad, and Bcl-xl were obtained from Cell Signaling Technology, and antibodies against Akt, p-Akt (Ser473), p-caspase 9, p-caspase 3, and cleaved PARP were obtained from Santa Cruz Biotech, Inc.