Subcellular localization of ␣ -fodrin in EoL-1 cells and peripheral blood eosinophils viewed by confocal microscopy. A , EoL-1 cells were stained with anti- ␣ -fodrin Ab ( right panel ) (magnification ϫ 128) and human eosinophils were stained with anti- ␣ -fodrin Ab ( left panel ) (magnification ϫ 192); nuclei in both panels were stained with DAPI. B , EoL-1 cells were double stained with anti- ␣ -fodrin (rhodamine-labeled secondary Ab) and F-actin-specific FITC-phalloidin. C , Flow cytometric analysis of EoL-1 cells stained with FITC-phalloidin for F-actin ( n ϭ 3; ء , p Ͻ 0.05 when compared with control (ANOVA) using Dunnet’s posthoc test). 

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... showed inhibition by LXA 4 , whereas the pSHP-2 blot showed significant synergistic stimulation by both GM-CSF and LXA 4 together. The lower small panels show the occurrence of the cytoplasmic marker GAPDH in the cytoplasmic fractions but not the membrane fractions. The observed fodrin translocation was consistent with immunofluorescence results (Fig. ...

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... fodrin is a known actin-associated protein, we further ex- amined the effects of GM-CSF and LXA 4 treatment on the cy- toskeleton by microscopy using anti-fodrin and F-actin FITC-phal- loidin to visualize F-actin. Fig. 6A clearly demonstrates the translocation of fodrin from the cytoplasm to the cell membrane after GM-CSF treatment. This translocation was prevented by pretreatment with LXA 4 , further validating our results shown in the Western blot of Fig. 5C. The experiment was repeated with human peripheral blood eosinophils, and the same results ...

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... the translocation of fodrin from the cytoplasm to the cell membrane after GM-CSF treatment. This translocation was prevented by pretreatment with LXA 4 , further validating our results shown in the Western blot of Fig. 5C. The experiment was repeated with human peripheral blood eosinophils, and the same results were obtained as shown in Fig. 6A. Further evidence of cytoskeletal rearrangement after GM-CSF treatment is given in Fig. 6B in which the poly- merized F-actin-specific FITC-phalloidin stain was employed. GM-CSF treatment of EoL-1 cells resulted in a dramatic rear- rangement of F-actin that was not restored by pretreatment with LXA 4 (Fig. 6B). LXA 4 alone had no ...

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... treatment. This translocation was prevented by pretreatment with LXA 4 , further validating our results shown in the Western blot of Fig. 5C. The experiment was repeated with human peripheral blood eosinophils, and the same results were obtained as shown in Fig. 6A. Further evidence of cytoskeletal rearrangement after GM-CSF treatment is given in Fig. 6B in which the poly- merized F-actin-specific FITC-phalloidin stain was employed. GM-CSF treatment of EoL-1 cells resulted in a dramatic rear- rangement of F-actin that was not restored by pretreatment with LXA 4 (Fig. 6B). LXA 4 alone had no effect on F-actin. FACS anal- ysis showed a 33% decrease in F-actin fluorescence intensity me- ...

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... same results were obtained as shown in Fig. 6A. Further evidence of cytoskeletal rearrangement after GM-CSF treatment is given in Fig. 6B in which the poly- merized F-actin-specific FITC-phalloidin stain was employed. GM-CSF treatment of EoL-1 cells resulted in a dramatic rear- rangement of F-actin that was not restored by pretreatment with LXA 4 (Fig. 6B). LXA 4 alone had no effect on F-actin. FACS anal- ysis showed a 33% decrease in F-actin fluorescence intensity me- diated by FITC-phalloidin in GM-CSF-treated samples (Fig. 6C). That the reduction in fluorescence was not prevented by LXA 4 was consistent with fluorescence microscopy results shown in Fig. 6B. Taken together, these ...

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... FITC-phalloidin stain was employed. GM-CSF treatment of EoL-1 cells resulted in a dramatic rear- rangement of F-actin that was not restored by pretreatment with LXA 4 (Fig. 6B). LXA 4 alone had no effect on F-actin. FACS anal- ysis showed a 33% decrease in F-actin fluorescence intensity me- diated by FITC-phalloidin in GM-CSF-treated samples (Fig. 6C). That the reduction in fluorescence was not prevented by LXA 4 was consistent with fluorescence microscopy results shown in Fig. 6B. Taken together, these results strongly suggested at least partial depolymerization of F-actin by ...

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... restored by pretreatment with LXA 4 (Fig. 6B). LXA 4 alone had no effect on F-actin. FACS anal- ysis showed a 33% decrease in F-actin fluorescence intensity me- diated by FITC-phalloidin in GM-CSF-treated samples (Fig. 6C). That the reduction in fluorescence was not prevented by LXA 4 was consistent with fluorescence microscopy results shown in Fig. 6B. Taken together, these results strongly suggested at least partial depolymerization of F-actin by ...

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... studies were conducted to inquire about fodrin and F- actin localization and organization after GM-CSF stimulation us- ing immunofluorescence microscopy. Since EoL-1 cells are quite small with large nuclei, we also included peripheral blood eosin- ophils for further confirmation in these studies. As shown in Fig. 6, GM-CSF stimulation of either EoL-1 cells or blood eosinophils resulted in the translocation of -fodrin to the plasma membrane. FACS analysis indicated substantial depolymerization of F-actin after GM-CSF stimulation that was not prevented by LXA 4 treat- ment (Fig. 6C). Furthermore, fluorescence microscopy using FITC-phalloidin showed ...

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... blood eosin- ophils for further confirmation in these studies. As shown in Fig. 6, GM-CSF stimulation of either EoL-1 cells or blood eosinophils resulted in the translocation of -fodrin to the plasma membrane. FACS analysis indicated substantial depolymerization of F-actin after GM-CSF stimulation that was not prevented by LXA 4 treat- ment (Fig. 6C). Furthermore, fluorescence microscopy using FITC-phalloidin showed considerable actin rearrangement after GM-CSF treatment that also was not prevented by LXA 4 treatment (Fig. 6B). These results gave strong evidence to support complex cytoskeletal involvement in the activation of eosinophils by GM- CSF and for the dysregulation of this ...

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... of -fodrin to the plasma membrane. FACS analysis indicated substantial depolymerization of F-actin after GM-CSF stimulation that was not prevented by LXA 4 treat- ment (Fig. 6C). Furthermore, fluorescence microscopy using FITC-phalloidin showed considerable actin rearrangement after GM-CSF treatment that also was not prevented by LXA 4 treatment (Fig. 6B). These results gave strong evidence to support complex cytoskeletal involvement in the activation of eosinophils by GM- CSF and for the dysregulation of this process by LXA 4. Our results were consistent with the occurrence of -fodrin modification(s) as a result of LXA 4 action that did not allow its association with the plasma ...