Increased proteolytic cleavage of lamin B and phosphorylation of c-jun after stimulation of the Fas receptor are tyrosine kinase-dependent events in human eosinophils. (A) Freshly isolated lavendustin A (16 μg/mL) -pretreated and untreated eosinophils were stimulated with anti-Fas MoAb for the indicated times. The cell lysates were analyzed by immunoblotting with an antilamin B MoAb. A proteolytic fragment of lamin B (arrowhead) was already present in freshly purified eosinophils. After 8 hours, a reduction in the amount of lamin B was observed in untreated but not in lavendustin A-pretreated cells. The intensity (od-Bkg/mm2) of the lamin B signal was analyzed by densitometry. (Upper panel) 1,787 (100%), 1,626 (91%), 1,881 (105%), 775 (43%), and 496 (28%). (Lower panel) 1,459 (100%), 1,551 (106%), 1,375 (94%), 1,618 (111%), and 1,156 (79%). The position of molecular size standards (left). (B) Lavendustin A abrogated phosphorylation of c-Jun. Freshly isolated lavendustin A (16 μg/mL) -pretreated and untreated eosinophils were stimulated with anti-Fas MoAb for 60 minutes. The cell lysates were immunoprecipitated with an anti-JNK Ab, and phosphorylation of GST-c-Jun (1-79) was observed by an in vitro kinase assay. No increase in phosphorylation of c-Jun was found in lavendustin A-pretreated eosinophils, suggesting that tyrosine kinase(s) activation is essential for Fas receptor-mediated JNK activation. All data are from at least three independent experiments.
