Cells grown in culture and retinal tissue were lysed in immunoblot buffer (0.5% SDS, 0.05 M Tris-HCl, pH 6.8, proteinase inhibitor cocktail, phosphatase inhibitor cocktail), homogenized, and protein concentration measured by Bradford colorimetric assay; 50 μg of lysate was resuspended in loading buffer (2% SDS, 1% dithiothreitol, and 0.05 M Tris-HCl, pH 6.8, 10% glycerol, 0.001% bromophenol blue), separated by 15% SDS-PAGE, and transferred to a nitrocellulose filter. The blots were blocked with PBS/5% nonfat dry milk for nonspecific binding and incubated with antibodies as follows: anti-Gadd45b (rabbit monoclonal, working dilution 1:10,000; Abcam, Cambridge, MA), anti-phospho-NFκB p65 (rabbit polyclonal, working dilution 1:200; Cell Signaling Technology, Danvers, MA), anti-NFκB (Cell Signaling Technology, rabbit polyclonal, working dilution 1:100), or anti-β-actin (mouse monoclonal, working dilution 1:10,000; Sigma-Aldrich, St. Louis, MO). Immunoblot assays were performed with peroxidase-conjugated goat anti-rabbit IgG2a or goat anti-mouse IgG and the enhanced chemoluminescence detection system (Amersham Life Science, Inc., Arlington Heights, IL) with β-actin used as an internal control. Each experiment was repeated at least three times.