(A) Western blot analysis using monoclonal antibodies CEACAM6 clone 9A6 and anti–β-actin. Ten micrograms of total protein from different intestinal epithelial cell lines were loaded onto 4%–12% Tris-glycine gel. (B) Confocal microscopic analysis of differentiated Caco-2 cells infected with GFP-expressing LF82 bacteria. Original magnification, ×400. CEACAM6 was detected using anti-CEACAM6 monoclonal antibody clone 9A6 and a Texas red–conjugated anti-mouse IgG (top panel). Arrows show colocalization (yellow) between CEACAM6 and bacteria. A 3D reconstruction (bottom panel) showed apical expression of CEACAM6 (red) and adherent bacteria (green). (C) Western blot analysis showing expression levels of CEACAM6, CEACAM5, and CEACAM1 by Caco-2 cells after 48 hours of stimulation with IFN-γ or TNF-α or after a 3-hour infection period with AIEC LF82 bacteria at an MOI of 10. As loading control, a labeling was performed using anti–β-actin polyclonal antibodies (D) Adhesion ability of AIEC LF82 bacteria was quantified after a 3-hour infection period at an MOI of 10 in Caco-2 intestinal epithelial cells after 1 and 2 days of IFN-γ stimulation. For RNA silencing, IFN-γ–stimulated Caco-2 cells were transfected with 10 ng of siRNA-blocking CEACAM6 (CEACAM6 siRNA) or 10 ng of nonworking siRNA (control siRNA). Expression of CEACAM6 was analyzed by Western blot analysis using anti-CEACAM6 monoclonal antibody clone 9A6 or anti–β-actin polyclonal antibodies. *P < 0.05 compared with nonstimulated Caco-2 cells; ^#P < 0.05 compared with Caco-2 cells stimulated for 2 days with IFN-γ and untransfected or transfected with control siRNA.