American Society of Hematology

Figure 7.

$Figure 7. Distribution and cytoskeletal association of CEACAM1-L in RBE cells. (A) Cell surface localization of CEACAM1-L during adhesion and spreading of serum-starved CEACAM1-L–transfected RBE cells on fibronectin (FN) and laminin-1 (LN) 75 minutes after plating. Unpermeabilized cells were stained with mAb Be9.2 and Cy-2–conjugated antimouse secondary antibody. Original magnification, × 400. (Bi) Translocation of CEACAM1-L into the Triton X-100–insoluble fraction during adhesion. The cells were solubilized with 0.5% Triton X-100 and insoluble CEACAM1-L was determined by immunoblotting of the Triton-insoluble fraction. β-Actin was used as a loading control. (ii) The amount of immunoblotted CEACAM1-L was quantified and is shown as the mean of 3 independent experiments ± SD. (C) Coimmunoprecipitation of talin with CEACAM1-L in spreading cells. Cells were kept in suspension (S) or were allowed to adhere for 75 minutes on fibronectin (FN) or laminin-1 (LN). Cells were solubilized and equal amounts of protein were immunoprecipitated with mAb Be9.2. Talin in complex with CEACAM1-L was detected by immunoblotting with mAb 8d4. Blots were reprobed with Be9.2 for detection of CEACAM1-L. A representative immunoblot of 3 independent experiments is shown. (D) Translocation of tyrosine double-mutant CEACAM1-L/Y448F/Y513F into the Triton X-100–insoluble fraction and coimmunoprecipitation of talin with CEACAM1-L/Y448F/Y513F during adhesion to fibronectin. Cells were solubilized 75 minutes after adhesion. CEACAM1-L/Y488513F in the pellet fraction was detected by immunoblotting with Be9.2. Talin in complex with the tyrosine double-mutant CEACAM1-L/Y488513F was detected in Be9.2-immunoprecipitated material with mAb 8d4. Representative immunoblots of 2 independent experiments are shown. IP indicates immunoprecipitation.$

Distribution and cytoskeletal association of CEACAM1-L in RBE cells. (A) Cell surface localization of CEACAM1-L during adhesion and spreading of serum-starved CEACAM1-L–transfected RBE cells on fibronectin (FN) and laminin-1 (LN) 75 minutes after plating. Unpermeabilized cells were stained with mAb Be9.2 and Cy-2–conjugated antimouse secondary antibody. Original magnification, × 400. (Bi) Translocation of CEACAM1-L into the Triton X-100–insoluble fraction during adhesion. The cells were solubilized with 0.5% Triton X-100 and insoluble CEACAM1-L was determined by immunoblotting of the Triton-insoluble fraction. β-Actin was used as a loading control. (ii) The amount of immunoblotted CEACAM1-L was quantified and is shown as the mean of 3 independent experiments ± SD. (C) Coimmunoprecipitation of talin with CEACAM1-L in spreading cells. Cells were kept in suspension (S) or were allowed to adhere for 75 minutes on fibronectin (FN) or laminin-1 (LN). Cells were solubilized and equal amounts of protein were immunoprecipitated with mAb Be9.2. Talin in complex with CEACAM1-L was detected by immunoblotting with mAb 8d4. Blots were reprobed with Be9.2 for detection of CEACAM1-L. A representative immunoblot of 3 independent experiments is shown. (D) Translocation of tyrosine double-mutant CEACAM1-L/Y448F/Y513F into the Triton X-100–insoluble fraction and coimmunoprecipitation of talin with CEACAM1-L/Y448F/Y513F during adhesion to fibronectin. Cells were solubilized 75 minutes after adhesion. CEACAM1-L/Y488513F in the pellet fraction was detected by immunoblotting with Be9.2. Talin in complex with the tyrosine double-mutant CEACAM1-L/Y488513F was detected in Be9.2-immunoprecipitated material with mAb 8d4. Representative immunoblots of 2 independent experiments are shown. IP indicates immunoprecipitation.

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