Acetylated p65

Cells were lysed in RIPA buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 1% NP40, 0.1% SDS) supplemented with a protease inhibitor cocktail (Calbiochem, Cambridge, MA, USA). The cells were then sonicated and centrifuged at 12,000 g for 10 min at 4°C to remove insoluble debris. The protein concentration was determined using the BCA assay ( Thermo Scientific, Waltham, MA, USA). Total proteins (30 µg) were resolved on a SDS-polyacrylamide gel, and the gels were then electroblotted onto a nitrocellulose membrane. After blocking with a 5% skim milk solution, the membranes were incubated with specific antibodies against acetylated p65 (1∶1,000; Cell Signaling Technology, Beverly, MA, USA) or β-actin (1∶5,000; Sigma-Aldrich, Hempstead, NY, USA), and the proteins were identified with the ECL detection system.

Primary keratinocytes were stimulated with the appropriate ligand for the time indicated in the legend. Media was aspirated and cells washed with ice cold PBS and harvested in Dundee lysis buffer38 (link). Lysates were analysed by SDS PAGE and probed with antibodies against p65, acetylated p65 (Cell Signalling Technology), Lamin B (Calbiochem) and GAPDH (Abcam). Secondary antibodies used were HRP-conjugated anti-mouse and anti-rabbit antibodies from Sigma. Blots were developed using enhanced chemiluminescence (ECL) reagents (Pierce).