Antibodies specific for caspase 3

Cells were lysed using RIPA buffer containing a protease inhibitor (Roche, Mannheim, Germany), and western blotting was performed with 20–30 μg of protein, according to standard procedures. The following primary antibodies were used for immunoblotting: c-Met (Invitrogen; 37-0100), CAPZA2 (Proteintech, Chicago, IL, USA; 15948-1-AP), HER2/ErbB2 (Thermo Scientific, Lab Vision; MS-730), MIEN1 (Abnova, Taipei City, Taiwan; Hooo84299-B01), GRB7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-607), and PGAP3 (Abcam, Cambridge, UK; ab81368). Antibodies specific for caspase-3 (Cell Signaling Technologies, Denver, MA, USA; #9662) and PARP (46D11, Cell Signaling Technology; #9532) were also used to detect apoptosis.

AY-27 cells were pretreated with EPI-H or EPI-AQ (as final concentration of epirubicin 0.25 (μg/mL) for 18 h. AY-27 cells were washed twice in cold PBS, and lysed in TBS containing 1 mM EDTA, 1 mM DTT, 0.2% Triton, 0.1% SDS, and the complete protease inhibitors mixture (Roche, Indianapolis, IN, USA). Proteins were subjected to SDS-PAGE, and then transferred to polyvinylidene difluoride membranes (Boehringer Mannheim, Indianapolis, IN, USA). Membranes were probed with antibodies specific for caspase-3 (Cell Signaling, Danvers, MA, USA), cleaved caspase-3 (Cell Signaling), or actin (Merck Millipore, Darmstadt, Germany), and incubated with the HRP-conjugated anti-rabbit Ig or anti-mouse Ig Ab. Signals were revealed with the ECL kit (Merck Millipore), and visualized by autoradiography. Increased levels of cleaved-caspase-3 (17 kDa) with a concomitant decrease in levels pro-caspase-3 (32 kDa) is regarded as an indicator of apoptosis.