Figure 2.

Endogenous SNO generation is iNOS dependent, and SNO is stable for 4 h following removal of LPS and rmIFN-γ. A) Duplicate wells of J774.2 cells were incubated in either fresh DMEM (unstimulated) or medium containing 1 μg/ml LPS and 1000 U/ml rmIFN-γ (stimulated) for 18 h. iNOS inhibitor 1400 W (100 μM) was also added to medium (stim.+1400W). Horizontal bars denote medians; n > 6. **P < 0.001; Mann Whitney U test. B) SNO concentrations of stimulated J774.2 cells were measured hourly following removal of LPS and IFNγ and replacement of medium with fresh DMEM. Lysates were produced using SNO-compatible lysis buffer plus 2% saponin. SNO content was determined by duplicate injection into I₃⁻ reaction mixture linked to ozone-based chemiluminescence and normalized to the protein content of each lysate. Data are presented as SNO concentration (pmol SNO/mg protein). Bars denote means + se; n > 6; ANOVA with Tukey’s multiple comparisons test.