Figure 5. CEACAM1 can be excluded from... | Rockefeller University Press

$Figure 5. CEACAM1 can be excluded from the CMD by adding tails from NHERF1-binding proteins. (A) GFP-CEACAM1–based constructs differ in their exclusion from the CMD 4 d after seeding. Images were collected with identical microscope settings to Fig. 1 C. (B) Cells were scored for GFP exclusion from the CMD as in Fig. 1 C. Only 5/169 cells appeared to exclude GFP-CEACAM1 from the CMD, and GFP-CEACAM1-DTHL (13/135), GFP-CEACAM1-PODXLΔ4 (10/237), and GFP-CEACAM1-CFTRΔ4 (12/148) were similar, whereas GFP-CEACAM1-PODXL (146/253) and GFP-CEACAM1-CFTR (94/111) were excluded from the CMD in most cells. (C) FRAP measurements indicated that GFP-CEACAM1 constructs that were excluded from the CMD were also less mobile in the apical membrane. (D) There was no significant difference between FRAP measurements of GFP-CEACAM1 and GFP-CEACAM1-DTHL after 120 s; however, there were significant differences between GFP-CEACAM1-PODXL and GFP-CEACAM1-PODXLΔ4, GFP-CEACAM1-CFTR and GFP-CEACAM1-CFTRΔ4, and GFP-CEACAM1-CBP and GFP-CEACAM1-CBPΔ4 (P < 0.005). (C and D) Each FRAP value is a mean of measurements from eight cells. Error bars represent standard deviation between measurements. (E) Cells expressing GFP constructs were grown on tissue culture plates and lysed when confluent. GFP constructs were immunoprecipitated from cell lysates, eluted, and Western blotted. Membranes were probed with anti-NHERF1 and either anti-GFP or anti-gp135 antibodies. The arrow to the left of the podocalyxin blot points to the GFP-PODXL band; the large band below is endogenous podocalyxin. GFP-PODXL and GFP-PODXLΔ4 lysate lanes represent 2% of the lysate used in the IP, and CEACAM1 lysate blots represent 1% of the lysate used in the IP. Molecular mass is indicated in kilodaltons. (F) Cells expressing high levels of GFP-CEACAM1-PODXL or GFP-CEACAM1-PODXLΔ4 were grown on filters for 4 d, methanol fixed, and stained for endogenous podocalyxin with anti-gp135 antibody. Podocalyxin did not appear to be excluded from the CMD in cells expressing GFP-CEACAM1-PODXL, whereas podocalyxin distribution was unaffected by expression of GFP-CEACAM1-PODXLΔ4. (G) CMD exclusion of endogenous podocalyxin was scored in cells expressing high levels of GFP-CEACAM1-PODXL or GFP-CEACAM1-PODXLΔ4 and in neighboring cells not expressing a GFP construct. Only 34/197 cells expressing GFP-CEACAM1-PODXL appeared to be excluding endogenous podocalyxin from the CMD, whereas 131/143 neighboring cells were excluding podocalyxin. The fraction of cells excluding podocalyxin was similar between those expressing GFP-CEACAM1-PODXLΔ4 (119/143) and neighbors (128/149). Images are projections of z stacks. Error bars represent standard deviation between experiments, except in D. Bars, 10 µm.$

Figure 5.

CEACAM1 can be excluded from the CMD by adding tails from NHERF1-binding proteins. (A) GFP-CEACAM1–based constructs differ in their exclusion from the CMD 4 d after seeding. Images were collected with identical microscope settings to Fig. 1 C. (B) Cells were scored for GFP exclusion from the CMD as in Fig. 1 C. Only 5/169 cells appeared to exclude GFP-CEACAM1 from the CMD, and GFP-CEACAM1-DTHL (13/135), GFP-CEACAM1-PODXLΔ4 (10/237), and GFP-CEACAM1-CFTRΔ4 (12/148) were similar, whereas GFP-CEACAM1-PODXL (146/253) and GFP-CEACAM1-CFTR (94/111) were excluded from the CMD in most cells. (C) FRAP measurements indicated that GFP-CEACAM1 constructs that were excluded from the CMD were also less mobile in the apical membrane. (D) There was no significant difference between FRAP measurements of GFP-CEACAM1 and GFP-CEACAM1-DTHL after 120 s; however, there were significant differences between GFP-CEACAM1-PODXL and GFP-CEACAM1-PODXLΔ4, GFP-CEACAM1-CFTR and GFP-CEACAM1-CFTRΔ4, and GFP-CEACAM1-CBP and GFP-CEACAM1-CBPΔ4 (P < 0.005). (C and D) Each FRAP value is a mean of measurements from eight cells. Error bars represent standard deviation between measurements. (E) Cells expressing GFP constructs were grown on tissue culture plates and lysed when confluent. GFP constructs were immunoprecipitated from cell lysates, eluted, and Western blotted. Membranes were probed with anti-NHERF1 and either anti-GFP or anti-gp135 antibodies. The arrow to the left of the podocalyxin blot points to the GFP-PODXL band; the large band below is endogenous podocalyxin. GFP-PODXL and GFP-PODXLΔ4 lysate lanes represent 2% of the lysate used in the IP, and CEACAM1 lysate blots represent 1% of the lysate used in the IP. Molecular mass is indicated in kilodaltons. (F) Cells expressing high levels of GFP-CEACAM1-PODXL or GFP-CEACAM1-PODXLΔ4 were grown on filters for 4 d, methanol fixed, and stained for endogenous podocalyxin with anti-gp135 antibody. Podocalyxin did not appear to be excluded from the CMD in cells expressing GFP-CEACAM1-PODXL, whereas podocalyxin distribution was unaffected by expression of GFP-CEACAM1-PODXLΔ4. (G) CMD exclusion of endogenous podocalyxin was scored in cells expressing high levels of GFP-CEACAM1-PODXL or GFP-CEACAM1-PODXLΔ4 and in neighboring cells not expressing a GFP construct. Only 34/197 cells expressing GFP-CEACAM1-PODXL appeared to be excluding endogenous podocalyxin from the CMD, whereas 131/143 neighboring cells were excluding podocalyxin. The fraction of cells excluding podocalyxin was similar between those expressing GFP-CEACAM1-PODXLΔ4 (119/143) and neighbors (128/149). Images are projections of z stacks. Error bars represent standard deviation between experiments, except in D. Bars, 10 µm.

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