Western blot and flow cytometric analysis of neutrophils treated with phorbol 12-myristate 13-acetate (PMA). (A) Lanes 1 to 3 represent neutrophils treated with 2 ng/ml PMA for 1, 2 and 5 h, respectively. Lane 4 (ANX1) is reference annexin 1. (B) Lane 1 represents neutrophils treated with 10 ng/ml PMA for 1 h and lane 2 represents neutrophils cultured in vehicle medium for 5 h. Each neutrophil lysate used for Western blot analysis contained 50 μg protein. Neutrophils from the same culture of panel A were also analyzed by flow cytometry (C). The results are representative of 8 experiments.