FICZ-induced enhancement of mediator release is calcium and ROS dependent. BMMCs were sensitized with E-C1 ± 1 nM FICZ or equal amount of vehicle as a control (Con) for 16 hours and then stimulated with 10 μg/mL OVA. The release of (A) Hex and (B) LTC4 in the absence or presence of a calcium blocker (2-APB) and an antioxidant (N-acetyl cysteine) was added 30 minutes before OVA stimulation. Data are representative of 3 independent experiments. (C) FICZ enhances oxidation of a PTP (SHP-2) in mast cells. BMMCs were sensitized with E-C1 ± 1 nM FICZ or an equal amount of vehicle as a control for 16 hours and then stimulated with 10 μg/mL OVA for the indicated time. The cells were lysed, and then SHP-1 and SHP-2 were immunoprecipitated (IP). The immunoprecipitates were subjected to alkylation by IAA, followed by reduction of the reversibly oxidized PTPs with dithiothreitol, and by final oxidation with pervanadate. After size fractionation by SDS-PAGE, the samples were subjected to immunoblotting (IB) with oxPTP mAbs and after stripping, with the respective Abs as loading controls. Data are representative of 3 independent experiments. (D) BMMCs were treated as above. OVA-stimulated cells were lysed and SHP-2 was immunoprecipitated, and the relative activity of PTPs was determined by measuring the levels of FDP fluorescence (RFU). *P < .05. Data are representative of 3 independent experiments.
