Cells were lysed in RIPA lysis buffer (Boston Bioproducts, Ashland, MA) and the resulting protein was quantified using the BCA protein assay kit (Thermo Fisher Scientific, Huntsville, AL). Cell lysates were mixed with NuPAGEH LDS 4X LDS sample buffer (Invitrogen) to 1X concentration; ten micrograms of total protein from each lysate was analyzed on a pre-cast NuPAGEH NovexH 10% polyacrylamide Bis-Tris gel (Invitrogen) together with the NovexH Sharp Pre-stained Protein Standard (Invitrogen) using NuPAGEH Tris-acetate SDS running buffer (Invitrogen). Protein from each gel was transferred to a polyvinylidene fluoride membrane using an iBlotH Dry Blotting System (Invitrogen) and 2X NuPAGEH transfer buffer (Invitrogen) containing 20% methanol and blocked with 3% bovine serum albumin (Sigma) in PBS pH 7.4 (Invitrogen). To detect CEA, each blot was probed overnight with 1 mg/mL of the CEACAM5-specific mAb clone 26/3/13 (Genovac), washed with PBS pH 7.4 containing 0.1% Tween-20, and subsequently probed for 1 hour with a horseradish peroxidase-conjugated donkey anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). After 5 washes with PBS pH 7.4 containing 0.1% Tween-20, each blot was exposed to SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and exposed to BioMax MS Kodak film (Sigma) for detection of chemiluminescence resulting from bound anti-CEACAM5 antibody. Equal amounts of protein loaded into each lane of the gel were controlled by detecting the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein using a rabbit anti-human GAPDH polyclonal antibody (Trevigen, Gaithersburg, MD) followed by a horseradish peroxidaseconjugated donkey anti-rabbit IgG (Jackson ImmunoResearch).