5 mM EDTA, and 1% Triton X-100) containing a protease inhibitor cocktail (Roche) for 30 min and centrifuged for 15 min at 21,300 g. The supernatant was used as the total cell extract. The protein concentration was determined using BCA Protein Assay Reagent (Pierce) Samples of 30 µg of total protein extracts reduced by 50 mM of sample reducing agent (RSA, Novex), were separated by SDS-PAGE (NuPAGE Novex TRIS acetate 3?8% gel, Invitrogen) and transferred to nitrocellulose membranes (Amersham Biosciences) The membranes were blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich) in 0.05% TBS-T (Tris-buffered saline supplemented with Tween-20) for 1 h at room temperature (RT), probed for 2 h with either an anti-HER2 rabbit polyclonal antibody (pAb-HER2, Neu (C-18):sc-284, Santa Cruz Biotechnology, dilution of 1:200) or a an anti-CEA rabbit monoclonal antibody (mAb-CEA, EPCEAR7 ab133633, Abcam, dilution of 1:1000), each diluted in blocking solution. Mouse monoclonal anti-?-actin antibody was used as protein loading control (mAb-?-actin, Abcam 8226, dilution of 1:1000) Blots were incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody, 300 mM NaCl GE Healthcare Life Sciences) and developed using chemiluminescent HRP substrate (Millipore Immobilon system). Signals were detected by using a Bio-Rad Chem-Doc luminescence detection system, p.8000 ,
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