Rabbit monoclonal anti phospho nfκb p65

Mouse monoclonal anti-NFκB p65, mouse monoclonal anti-CaMKII, rabbit polyclonal anti-BDNF, and rabbit polyclonal anti-integrin αM antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse monoclonal anti-actin, mouse monoclonal anti-NR2B and rabbit polyclonal anti-NR2A antibodies were purchased from Millipore (Billerica, MA, USA). Mouse monoclonal anti-GFAP, rabbit monoclonal anti-phospho-NFκB p65, rabbit monoclonal anti-TNF-α, rabbit monoclonal anti-IL-6, mouse monoclonal anti-IL-1β, and horseradish peroxidase (HRP)-conjugated IgG antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). ECL Western Blotting Reagents were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cells were harvested, washed once with ice-cold PBS, and lysed with RIPA buffer [150 mM NaCl, 1% NP-40, 50 mM Tris–HCl, pH 7.5, 0.1% SDS, and 0.5% sodium deoxycholate supplemented with 1 × protease inhibitor cocktail (Roche Applied Science)]. Protein concentrations were determined by a bicinchoninic acid protein assay kit (Novagen). Protein samples were run on a 12% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore) at 100 V for 2 h. Membranes were blocked in 5% skimmed milk and then incubated with primary antibody at 4°C overnight. Membranes were washed three times for 15 min each with Tris-buffered saline, 0.1% Tween 20 buffer. A horseradish peroxidase-conjugated secondary antibody was incubated with the membrane for 1 h and followed by three washes as mentioned above. The membranes were developed by the SuperSignal West Femto developing reagent from Pierce Biotechnology. Monoclonal or polyclonal Abs used were: goat monoclonal anti-β-actin from Santa Cruz Biotechnology; rabbit polyclonal anti-IRAK1, rabbit monoclonal anti-TRAF6, rabbit monoclonal anti-NF-κB p65, and rabbit monoclonal anti-phospho-NF-κB p65 from Cell Signaling Technology.