Nef-treated microglial cells release iNOS-dependent neurotoxic factors.

<p>(<b>A</b>) BV-2 cells were treated for 48 h with myr⁺Nef_SF2 (200 ng/ml) in the presence (light gray bars) or absence (dark gray bars) of L-NAME at 10 μM concentration. Supernatants were collected and NO₂^- content was measured using the Griess colorimetric assay. (<b>B</b>) iNOS expression was evaluated on total cell lysates by Western Blot using specific anti-iNOS antibodies (upper panel) and anti-β-tubulin antibodies as an internal loading control (lower panel). (<b>C</b>) NB41A3 cells were incubated for 24 h with supernatants from (<b>A</b>). (<b>D</b>) NB41A3 cells were treated for 24 h with myr⁺Nef_SF2 (200 ng/ml) or with LPS (100 ng/ml). Black line: No L-Name treatment; Gray line: L-Name (10 μM). (<b>E</b>) NB41A3 cells were incubated for 24h with conditioned supernatants derived from untreated BV-2 cells or treated for 48 h with 200 ng/ml of wild type (WT), G2A, 4EA’, 4EA” and ΔN-terminal deleted myr⁺Nef_SF2 proteins. In (<b>C)</b> to (<b>E)</b>, cell viability was measured by flow cytometry using Propidium Iodide (PI) dye exclusion and data were expressed as percentage of PI positive cells. One out of three independent experiments is shown.