Detection of F-actin in NGF-stimulated PMCs with or without pretreatment with C botulinum C2 toxin.
(A) PMCs were allowed to migrate toward 50 ng/mL NGF for 1 hour following pretreatment with (right panel) or without (left panel) 300 ng/mL C botulinum C2 toxin for 2 hours. PMCs were fixed, permeabilized, and stained with Oregon Green 488-phalloidin. F-actin was visualized using a confocal laser scanning microscope (objective × 2000). The mast cell stimulated with NGF shows increased fluorescent intensity that is concentrated in lamellipodia and other submembranous sites (left panel). In contrast, the mast cell pretreated with C botulinum C2 toxin before the addition of NGF is rounded and shows a very weak, positive reaction for F-actin (right panel). (B) PMCs were treated with 50 ng/mL NGF for 2 hours following pretreatment with or without 300 ng/mL C botulinum C2 toxin for 2 hours and then analyzed by flow cytometric assay.
