Fig. 5

NF-κB localization in co-cultured BMEC, astrocytes, and microglia. a PE-CAM (green) and p65 NF-kB (red) immunofluorescence in WT BMEC that were seeded into transwell inserts and treated with PBS or IL-1β (50 ng/mL) for 30 min. Blue = DAPI. Scale bar = 50 μm. Arrows indicate examples of PE-CAM-positive cells with concentrated nuclear NF-κB expression. b GFAP (blue), YFP (green), and p65 NF-κB (red) immunofluorescence in CX3CR1-YFP-Cre mixed glia that were seeded into lower transwell chambers containing PBS- or IL-1β-treated BMEC in the upper inserts. Scale bar = 50 μm. Arrows indicate nuclear expression of NF-κB in YFP-labeled cells. c Percentage of BMEC and microglia with nuclear NF-κB localization. ***p < 0.001 vs. PBS-treated BMEC. d Luciferase activity in NF-κB Luc SIM-A9 cells that were co-cultured with WT BMEC in transwells. PBS or IL-1β (50 ng/mL) was added to both the upper and lower chambers for 6 h. NF-κB Luc SIM-A9 cells were also treated with PBS, IL-1β (50 ng/mL), PBS + 0.1% BSA, or LPS (10 ng/mL) for 6 h in the absence of BMEC. Luminescence was normalized to total protein content. n = 4 per group. Data are expressed as mean ± SEM. ***p < 0.001 vs. all other groups