Nef myristoylation and the acidic cluster are essential to induce iNOS.

<p>BV-2 cells were left untreated or incubated with 100 ng/ml wild type (WT), G2A, two different preparations of the EEEE→AAAA mutant (4EA’ and 4EA”) and ΔN-terminal deleted myr⁺Nef_SF2 proteins. (<b>A</b>) iNOS mRNA was measured by real time RT-PCR using total RNA isolated from cells treated for 6 h. (<b>B</b>) Total cellular extracts obtained from cells treated for 24h were analyzed by Western Blot using anti-iNOS antibodies, whereas β-tubulin expression levels were used as an internal loading control. (<b>C</b>) Supernatants collected from cells treated as in (<b>B</b>) were measured for NO₂^- content using the Griess colorimetric assay. One out of three independent experiments is shown.