Western Blot Analysis of Neuroinflammation

The brain tissues, treated astrocytes, and microglial BV-2 cells were prepared as previously described (Gu et al. 2015 (link)). An equal amount of total protein (20 µg) was resolved on 8–15% sodium dodecyl sulfate polyacrylamide gel and then transferred to a PVDF membrane (Immobilon-P; pore size 0.45 µm, EMD Millipore, USA). The membranes were blocked for 1 h in 5% skim milk solution and incubated for overnight at 4 °C with specific antibodies. To detect target proteins, specific antibodies against APP, iNOS (1:1000, Novus Biologicals, Inc., Littleton), BACE1, IBA-1 (1:1000, Abcam, Inc., Cambridge, MA, USA), COX-2 (1:1000, Cell Signaling Technology, Inc., Beverly, MA, USA), GFAP, p50, p65, IκB, phospho-IκB, β-actin, Histone H1 (1:1000, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) were used. The blots were then incubated with one of the following corresponding conjugated secondary antibodies: goat anti-rabbit, goat anti-mouse, or donkey anti-goat IgG-HRP (1:5000; Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA). Immunoreactive proteins were detected with an enhanced chemiluminescence Western blotting detection system. The relative density of the protein bands was scanned by densitometry using MyImage (SLB, Seoul, Korea) and quantified by Labworks 4.0 software (UVP Inc., Upland, CA, USA).