Figure 3

AAT effects on α-spectrin cleavage in neutrophils. Neutrophils were cultured alone or exposed to AAT for 18 h. Equal amounts of cell lysates were separated on 6.5% SDS-PAGE after Western blot analysis. Blots were immunostained with an anti-human α-spectrin monoclonal antibody (1:2,000 dilution) followed by incubation with horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody diluted 1:10,000. The enhanced chemiluminescence kit was used to visualize immunolabeling. A 145-kDa calpain-mediated and 150-kDa (calpain- and caspase 3-mediated) fragments are clearly detectable in controls cells. The 145-kDa calpain-mediated fragment is not detectable in neutrophils treated with AAT. Weak staining for the 150-kDa (calpain- and caspase 3-mediated) fragment is present. A representative Western blot is shown (n = 3).