Anti phospho ikkα β

Cells were dissociated with PBS + 3 mmol/L ethylenediamine-tetraacetic acid and lysed in a buffer containing 50 mmol/L Tris, 150 mmol/L NaCl, 5 mmol/L ethylenediaminetetraacetic acid, 1% Triton X-100 (all from Sigma), and protease inhibitors (Thermo Fisher Scientific). Protein concentrations were determined using a Bio-Rad protein assay (Bio-Rad) with BSA as the standard. Samples were denatured in Laemmli buffer (Bio-Rad) with βME (2-mer-captoethanol, Bio-Rad) at 95°C for 5 minutes. Cell lysates (5 μg/lane) were run on a 10% SDS polyacrylamide gel and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 5% BSA (for phosphorylated proteins) or 5% milk in Tris-buffered saline + 0.1% Tween-20 (all from Sigma) and then probed with either anti–phospho-IKKα/β (Cell Signaling Technology), anti-IKKα (Cell Signaling Technology), or anti-HA (Bethyl Laboratories, Inc.). Anti-rabbit IgG, HRP-linked or anti-mouse IgG, HRP-linked was used as secondary antibody (Cell Signaling Technology). GAPDH or β-actin (Santa Cruz Biotechnology) was used as a housekeeping gene. Blots were developed using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and exposed to GeneMate Blue Basic Autoradiography Film (BioExpress).