Western blot analysis of iNOS content in cytokine-treated macrophage J774 cells. Cytoplasmic extracts were prepared from murine J774 macrophages untreated and treated with IFN-γ/LPS for 24 hours as described in Fig 1. Equal amounts (50 μg) of denatured proteins were electrophoresed in an SDS 10% polyacrylamide gel, electroblotted to filters, and incubated with anti-iNOS antibody (1:1,000 dilution) followed by secondary antibody as described in Materials and Methods. Bands were visualized by chemiluminescence. Migration of molecular mass markers (myosin, phosphorylase B, and glutamic dehydrogenase, 250, 148, and 60 kD, respectively), loaded on the same gel, is shown on the left. The results shown are representative of six separate experiments.