Colocalization and molecular association of PR3 and hPLSCR1 in neutrophils. (A) Immunofluorescence microscopy analysis of neutrophils after PR3 (left) and hPLSCR1 (right) immunolabeling (magnification × 63). (B) Confocal microscopy analysis of indirect fluorescence immunolabeling of PR3 (green) and hPLSCR1 (red) under basal conditions and after physiologic apoptosis induction. Images were obtained as described in Figure 2. Under resting conditions, punctuated membrane PR3 labeling was easily detected in SLO-permeabilized neutrophils. hPLSCR1-immunofluorescence patterns were similar to those observed for PR3. The merged fluorescence (white) showed cytoplasmic and membrane colocalizations of PR3 and hPLSCR1 in resting and apoptotic neutrophils. Control experiments with mouse or rabbit IgG antibodies alone were not labeled (data not shown). (C) Percentages of PR3-hPLSCR1 colocalizations calculated with LSM software. PR3 labeling colocalized with the hPLSCR1 (**P < .01) based on counts of 401 basal and 112 apoptotic neutrophils from 5 different donors. Data are mean (± SEM). (D,E) Western blots of reciprocal coimmunoprecipitation from total neutrophil lysates (TL). Immunoprecipitates (IP) were analyzed by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes. PR3 was detected as a single band at 30 kDa in TLs and in both PR3 (IP PR3) and hPLSCR1 (IP SCR) immunoprecipitates by a rabbit polyclonal anti-PR3 (D); hPLSCR1 was detected as a single band at 37 kDa in TLs and in both hPLSCR1 (IP SCR) and PR3 (IP PR3) immunoprecipitates by a rabbit polyclonal anti-hPLSCR1 (E). TLs were also immunoprecipitated with mouse (IP mIgG) or rabbit (IP rIgG) control IgG and no PR3 or hPLSCR1 was detected. Vertical lines have been inserted to indicate a repositioned gel lane.