Inhibition of iNOS does not block the TNF-α+IFN-γ-stimulated increases in levels of endogenous APP, BACE1, or secreted Aβ40 in mouse primary astrocyte cultures. (A) Cell lysates of cytokine-stimulated mouse primary astrocytes analyzed in Fig. 1A-C were prepared for iNOS (NOS2) immunoblot. The 130 kDa iNOS band is indicated by the arrowhead. Pro-inflammatory agents (alone and combinations) and stimulation times are shown. GFAP immunosignal served as a loading control. Note that iNOS levels were more strongly induced in astrocytes that were stimulated by pro-inflammatory agent combinations but not by single agent treatments, with the exception of LPS. (B-G) Cultured wild-type C57BL/6J mouse primary astrocytes were pre-treated for 30 min with the iNOS inhibitor 1400 W at 0, 8, 25, or 50 μM and were then stimulated for 96 h with TNF-α+IFN-γ. Cell lysates and CMs were harvested and analyzed for endogenous levels of iNOS (B), nitrite production in CM (C), APP (D, E) and BACE1 (D, F) by immunoblot and secreted Aβ40 by ELISA (G). β-actin or GFAP immunosignals served as loading controls. Histograms in E-G represent quantifications of signals for APP (E) and BACE1 (F), as well as that of secreted Aβ40 (G) expressed as percent of un-stimulated vehicle control. TNF-α+IFN-γ treatment alone significantly elevated astrocytic APP, BACE1, and secreted Aβ40 levels over un-stimulated vehicle controls ("0" bars in E-G). Note that iNOS inhibition did not significantly block the TNF-α+IFN-γ-stimulated increases in levels of astrocytic APP, BACE1, or secreted Aβ40. Error bars, SEM (n = 3).