rPLSCR1 expression in RBL-2H3 lines expressing recombinant PR3 or its inactive PR3S203A mutant. (A) Intracellular expression of PR3 and PLSCR1 in RBL/CT, RBL/PR3, and RBL/PR3S203A. Flow-cytometry analysis of intracellular expression of PR3 in RBL transfectants. (B) Western-blot analysis of PLSCR1 (using the mouse monoclonal 129.2) in lysates under resting conditions or after etoposide-induced apoptosis. RBL cells were lysed in lysis buffer and 10 μg of protein was loaded/well. (C) Double immunofluorescence labeling of PR3 and rPLSCR1 in RBL/PR3 and RBL/PR3S203A after SLO permeabilization. The merged fluorescence showed a membrane PR3-rPLSCR1 colocalization under resting conditions. Images were obtained as described in Figure 2. (D) Flow-cytometry analysis of rPLSCR1 membrane expression (bold line) compared with the isotypic control (thin line) in RBL/CT, RBL/PR3, and RBL/PR3S203A after etoposide-induced apoptosis (top panels). Double labeling of annexin-V and PLSCR1 is shown in the bottom panels. A representative experiment of 4 different experiments giving the same result is shown.