Inhibition of JAK blocks the TNF-α+IFN-γ-stimulated increase in endogenous APP level in mouse primary astrocytes, but not that of BACE1 or secreted Aβ40. Cultured wild-type C57BL/6J mouse primary astrocytes were pre-treated for 30 min with JAK Inhibitor I at 0, 1, 5, and 20 μM and then stimulated for 96 h with TNF-α+IFN-γ. Cell lysates and CMs were harvested and analyzed for endogenous levels of APP (A, B) and BACE1 (A, C) by immunoblot and secreted Aβ40 by ELISA (D). (A) Immunoblot images for APP, BACE1, and GFAP (loading control) signals. Lanes with lysates of astrocytes that received inhibitor treatments and TNF-α+IFN-γ stimulation are indicated. (B-D) Histograms represent quantifications of signals for APP (B) and BACE1 (C), as well as that of secreted Aβ40 (D) 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 B-D). In contrast, JAK Inhibitor I significantly reduced the TNF-α+IFN-γ-stimulated increase in astrocytic APP level ("20" bar in B; #: p < 0.05, n = 3). Statistical analysis was the same as described in Fig. 1. Error bars, SEM.