Rabbit antienf kb p65

Cells were lysed in RIPA lysis buffer (Cell Signaling Technology, Danvers, MA), normalized, subjected to SDS-PAGE, and blotted onto polyvinylidene fluoride membranes. After blocking in 5% milk and/or tris-buffered saline with Tween 20, membranes were probed with the indicated antibodies and visualized with horseradish peroxidaseeconjugated secondary antibodies using ECL Substrate (Bio-Rad, Hercules, CA). The following primary antibodies were used: mouse anti-actin antibody from Sigma; rabbit anti-IL17RB from Proteintech; rabbit anti-pS6 (S235/6), rabbit antiephosphorylated protein kinase B (S473), rabbit antiephosphorylated extracellular signaleregulated kinase 1/2 (T202/Y204), rabbit antiephosphorylated NF-kB p65, rabbit antieNF-kB p65, rabbit anti-S6, rabbit antieprotein kinase B, mouse antieextracellular signaleregulated kinase 1/2, mouse anti-STAT3, rabbit antiephosphorylated STAT3 (S727), and rabbit antiephosphorylated STAT3 (Y705) from Cell Signaling Technology; and rabbit anti-IL17RA from Abcam. ImageJ software was used to quantify the intensity of nonsaturated western blot bands. For mesoscale, RHE was lysed in RIPA buffer and IL-17E was measured using the U-PLEX Human IL-17E/IL-25 Assay set according to manufacturer's protocol (Meso Scale Diagnostics, Rockville, MD).

Paraffin-embedded sections (7-mm thick) were examined using the following primary antibodies: mouse anti-NF200
(1:400; Sigma), mouse antieglial fibrillary acidic protein (GFAP) (50 mg/mL; Progen, Heidelberg, Germany), goat anti-iba1 (1:400; Abcam, Cambridge, England), goat anti-TLR4 (2 mg/mL; sc-16240; Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit antieNF-kB p65 (1:500; catalog number 8242; Cell Signaling, Danvers, MA). The antibodies against TLR-4 18,24,30,31 and NF-kB 32, 33 have been widely used and described elsewhere. Stained sections were examined using a microscope (BX51; Olympus Corporation, Tokyo, Japan) connected to a DP70 camera (Olympus). Images were processed and viewed using DP manager software version 2.2. 1.195 (Olympus). Quantitative analysis of the stained region was performed using ImageJ software version 1.46r (NIH, Bethesda, MD; http://imagej.nih.gov/ij).