Induction of iNOS accumulation in LPS/IFN-γ–treated monocytes. Cytosolic extracts were prepared from untreated monocytes and from monocytes and murine J774 macrophages treated with cytokines for 24 hours as described in Fig 1. Proteins (100 μg) of treated and untreated monocytes and 25 μg of J774 cells were analyzed on SDS 10% polyacrylamide gels and blotted to filters that were incubated with primary (anti-iNOS, 1:500 dilution) and 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. Similar results were obtained in all the experiments on cytokine stimulation of both monocytes and monocyte-derived macrophages. Accumulation of iNOS was detected in human monocytes after LPS/IFN-γ stimulation, although to a lower extent than in mouse J774 macrophages.