Mouse monoclonal against β actin

Cells were harvested, sonicated, and centrifuged. Supernatants were used for immunoblotting. Samples (20–30 ug) were fractionated by SDS PAGE and electroblotted onto nitrocellulose membranes, and membranes were blocked in Tris-buffered saline, pH 7.6, containing 0.5% Tween-20 and 5% nonfat milk. Primary antibodies p-NF-κB p65 (S536) (93H1) rabbit monoclonal (1:600; Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal against p65 NF-κB (1:1000; Abcam), rabbit polyclonal against GFAP (1:1000; Dako, Carpinteria, CA, USA), and mouse monoclonal against β-actin (1:3000; Abcam) were applied overnight at 4°C followed by secondary antibodies goat antirabbit IgG (heavy chain + light chain) − horseradish peroxidase conjugate (1:5000; Bio-Rad) and goat antimouse IgG (heavy chain + light chain) − horseradish peroxidase conjugate (1:5000; Bio-Rad) for 90 minutes at room temperature. After washing, the immunoblots were visualized by chemiluminescence.