Characterization of wild-type and CEACAM1-L–transfected rat brain endothelial (RBE) cells. (A) Immunohistochemistry of vascular endothelia in rat meninges using rat CEACAM1-specific monoclonal antibody Be9.2 or isotype control IgG (insert in the upper left); representative staining of a cross-section is shown. Original magnification, × 10. (B) Expression of VE-cadherin and PECAM-1 in RBE cells. Immunoblotting of cell lysates (50 μg) of (lane 1) IEC-6 (small intestine epithelial cells, negative control) or (lane 2) RBE cells. (C) Flow cytometric detection of E-selectin on the surface of RBE cells. Isotype control IgG staining of untreated cells (gray shading), anti–E-selectin staining of untreated cells (–), or 20-hour TNF-α–treated cells (—). (D) Detection of CEACAM1 mRNA by RT-PCR in wild-type (wt) and CEACAM1-L–transfected RBE cells (CEACAM1-L); the primer combinations recognized all isoforms of rat CEACAM1 (L/S), the long isoform specifically (L), or the short isoform specifically (S). (E) Cell surface expression of CEACAM1-L after transfection. Wt (top) or CEACAM1-L–transfected and cloned cells were incubated with isotype control (gray shading) or mAb Be9.2 (—) and FITC-coupled secondary antibody for flow cytometric analysis. (F) Detection of soluble and membrane-bound CEACAM1-L. Immunoprecipitated soluble isoforms of CEACAM1 from cell culture supernatants (top) or membrane-bound CEACAM1 (bottom) of rat RBE and MTC cells detected by immunoblotting with mAb Be9.2. A representative immunoblot of 3 independent experiments is shown.