CEACAM1 can be excluded from the CMD independently of NHERF1 by adding domains that bind more directly to the cytoskeleton. (A) Addition of NHERF1’s ERM-binding domain or ezrin’s actin-binding domain is sufficient to exclude GFP-CEACAM1 from the CMD 4 d after seeding. NHERF1 was knocked down in GFP-CEACAM1-NHERF1 cells (GFP-CEACAM1-NHERF1 shRNA), and exclusion was similar to control cells (GFP-CEACAM1-NHERF1 vector). Images were collected with identical microscope settings to Fig. 1 C. (B) GFP-CEACAM1-NHERF1 shRNA and GFP-CEACAM1-NHERF1 vector cell lysates were blotted with anti-NHERF1 and antiezrin antibodies. Molecular mass is indicated in kilodaltons. (C) Cells were scored for GFP exclusion from the CMD as in Fig. 1 C. 113/199 cells expressing GFP-CEACAM1-NHERF1 appeared to exclude the construct from the CMD, and similar results were seen for GFP-CEACAM1-ezrin (105/185). After shRNA knockdown of NHERF1, GFP-CEACAM1-NHERF1 was excluded from the CMD in 193/393 cells, a similar ratio to cells only treated with vector (125/232). Error bars represent standard deviation between experiments. (D) FRAP measurements indicated that GFP-CEACAM1 was less mobile in the apical membrane after addition of an ERM- or actin-binding domain, and this was independent of NHERF1 knockdown. (E) There are significant differences in the FRAP measurements between GFP-CEACAM1 and both GFP-CEACAM1-NHERF1 (**, P < 0.005) and GFP-CEACAM1-ezrin (*, P < 0.05) after 120 s. No significant difference was seen in the FRAP of GFP-CEACAM1-NHERF1 after shRNA knockdown of NHERF1. GFP-CEACAM1 data are from Fig. 5 and are included as a reference. (D and E) Each FRAP value is a mean of measurements from eight cells. Error bars represent standard deviation between measurements. Images are projections of z stacks. Bars, 10 µm.