Combinations of pro-inflammatory cytokines elevate endogenous BACE1 levels in mouse primary astrocyte cultures. (A, B) Cell lysates of cytokine-stimulated mouse primary astrocytes analyzed in Fig. 1A-C were analyzed for BACE1 by immunoblot. (A) BACE1 immunoblot images. The mature BACE1 band at 70 kDa is indicated by the arrowhead. Pro-inflammatory agents (alone and combinations) and stimulation times are indicated. Cell lysate from un-stimulated BACE1-/- primary astrocytes was used as a negative control, while cell lysate from a stable BACE1-overexpressing HEK-293 cell line was used as a positive control. GFAP and β-actin immunosignals were loading controls as in Fig 1A-C. (B) Histograms represent quantifications of BACE1 immunoblot signals in (A) expressed as percent of vehicle control. Note that cytokine combinations including TNF-α and IFN-γ were generally more potent at increasing endogenous BACE1 levels in astrocytes over time in culture, raising BACE1 levels to ~600% of control. (C) mRNAs prepared from cytokine-stimulated primary astrocytes in Fig 1D were analyzed for endogenous BACE1 mRNA levels by TaqMan quantitative RT-PCR. Histograms represent quantifications of BACE1 mRNA levels expressed as percent of vehicle control. Note that astrocytic BACE1 mRNA levels were significantly reduced by TNF-α+IFN-γ stimulation (C), even though BACE1 protein levels were increased several fold in similarly treated astrocytes (B). Statistical analysis was the same as described in Fig. 1. Significance indicates comparison to individual vehicle control within each measurement and each time point (n = 2-3; *p < 0.05, **p < 0.01, ***p < 0.001). Error bars, SEM.