Human peripheral monocytes but not neutrophils (polymorphonuclear cells) express the annexin A2 heterotetramer. (A) Western blotting. Monocyte and neutrophil cell (PMN) lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against annexin A2, S100A10, and α-actin as control. (B) Flow cytometry. Cells were stained with control rabbit or mouse IgG (open histograms), annexin A2 and S100A10 antibodies (filled histograms). Primary antibodies were visualized by PE-conjugated IgG F(ab)2 and cells were analyzed by FACScan. CD14 and CD47 served as controls. (C) Immunofluorescence microscopy. Monocytes were fixed and stained with mouse antibody against S100A10 followed by anti-mouse rhodamine red-labeled IgG F(ab)2, and rabbit anti-annexin A2 antibody followed by anti-rabbit FITC-labeled IgG F(ab)2. Cells were analyzed by fluorescence microscopy. Overlay picture shows colocalization of annexin A2 and S100A10. Immunostaining for annexin A2 and S100A10 was not imitated by antibodies against p38 MAPK or actin (data not shown). (D) Coimmunoprecipitation. S100A10 and annexin A2 were immunoprecipitated from monocyte lysates using anti-S100A10 antibodies and protein A/G agarose. Immunoprecipitates were analyzed by immunoblotting using mAbs against annexin A2 and S100A10. Input is the loading control, that is, cell lysate. IgG indicates negative control; IP, immunoprecipitation. HC and LC indicate IgG heavy and light chain, respectively. PAR1 immunostaining served as control. (E) Localization of the annexin A2 heterotetramer at the monocyte surface. Monocytes were surface biotinylated and lysed. The biotinylated proteins were precipitated with immobilized neutravidin and the cross-linker was removed by DTT. From these recovered surface proteins, annexin A2 and S100A10 were coimmunoprecipitated using anti-S100A10 mAbs and analyzed by Western blotting. Loading control (input, lane 1) and the immunoprecipitate (lane 2) were stained using mAbs against annexin A2 and S100A10. PAR1 immunostaining was used as control. IP indicates immunoprecipitation. All results are representative of at least 3 experiments.
