Fig. 2. FLNa directly binds to CEACAM1-L. (A) Affinity precipitation of purified CC1-cyto-His or His-tagged control protein by purified GST or GST-FLNa (aa 2460-2647) bound to glutathione-Sepharose. Associated CC1-cyto-His or control-His was detected by mAb anti-Xpress. (B) SPR binding kinetics of GST-FLNa to immobilized CC1-cyto-His. Representative overlaid sensograms illustrating the real-time binding of GST-FLNa at various concentrations (42, 84, 126 and 168 nM from bottom to top) to CC1-cyto-His immobilized on a CM5 chip with substracted background are shown. The inset shows the primary sensogramms of the flow cells with immobilized CC1-cyto-His and immobilized control protein (background) after injection of 168 nM GST-FLNa. RU, relative units. (C) Affinity precipitation of CEACAM1-L from lysates of CEACAM1-L-transfected rat brain endothelial cells (RBE) by purified GST or GST-FLNa bound to glutathione-Sepharose. Associated CEACAM1-L was detected by rat CEACAM1 specific mAb Be9.2. (D) RBE cell lysate and rat liver lysate were immunoprecipitated with mAb Be9.2 or an Ig-control. CEACAM1 was detected with mAb Be9.2. Co-precipitated FLNa was detected by mAb FLMN01. (E) Cell lysates from human colon carcinoma HT29 cells or freshly prepared and PMA-treated human granulocytes were immunoprecipitated with human CEACAM1 reactive mAb 4/3/17 or Ig-control. CEACAM1 was detected with mAb 4/3/17. Coprecipitated FLNa was detected with mAb FLMN01.