FIGURE 3.

Numerous PTPs are oxidized in the course of mast cell activation. A and B, IgE-sensitized RBL cells (A) or BMMCs (B) were nonactivated (Control) or activated with 5 mm H₂O₂, 0.2 mm Pv, or TNP/BSA (Ag; 0.5 μg/ml). After 15 min, the cells were solubilized in lysis buffer containing 0.2% Triton X-100. Postnuclear supernatants were size-fractionated by SDS-PAGE and analyzed by immunoblotting (IB) with mAb specific for the oxidized PTP active site. After stripping, the membranes were probed with anti-SHP-1 antibody as a loading control (SHP-1). Numbers on the left and right indicate, respectively, position of molecular mass markers (in kDa) and localization of phosphatases as identified by immunoprecipitation and/or mass spectrometry (see supplemental Fig. S2 and Tables S1 and S2). C, RBL cells were activated with 1.6 mm H₂O₂ for the indicated time intervals. The cells were lysed and processed as in A. D, RBL cells were activated for 40 min with different concentrations of H₂O₂. Subsequently, the cells were lysed in 0.2% Triton X-100 and processed as in A. E, RBL cells were first lysed with 0.2% Triton X-100, and the postnuclear supernatants were exposed to H₂O₂ at the indicated concentrations. After 40 min, the lysates were fractionated by SDS-PAGE and analyzed as in A. F, BMMCs were activated for 40 min with different concentrations of H₂O₂. Subsequently, the cells were lysed in 0.2% Triton X-100 and processed as in A. Typical experiments from at least three performed are shown.