Eight weeks old F2 pups (one human CEACAM1 gene positive and one negative by PCR) were euthanized by CO 2 inhalation. Liver, heart, lung, brain, kidney, spleen, small intestine (ileum), and neutrophil (from peripheral blood) or fecal pellets were homogenized and lysed in 1% NP-40 lysis buffer. Cell lysates were incubated on ice for 30 min, followed by centrifugation for 30 min at 4uC at 14000 rpm. Total protein (50 mg) was separated by sodium dodecyl sulfate-gel polyacrylamide electrophoresis, transferred to nitrocellulose membranes and probed with either anti-murine CEACAM1 mAb CC1 (1 mg/mL, kind gift from Dr. Kathryn Holmes, University of Colorado health Sciences, Denver, CO) and anti-b-actin antibody (1 mg/mL, Abcam, Cambridge, UK) antibody or anti-human CEACAM1 mAb T84.1 (1 mg/mL) [32] . Signals were detected on the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).