Antiphospho iκb α antibody

The cells were twice washed with cold PBS and collected by scraping after PRO-PREPTM (Intron Biotechnology, Seongnam, Korea). Then the cells were lysed in ice for 30 minutes with lysis buffer. The lysates were clarified by centrifugation in 12,000 rpm, 30 minutes at 4°C and the supernatant was collected. The protein concentration was determined using the Bradford assay. Then 30 μg of protein from each sample were boiled for 5 minutes and separated by SDS-PAGE using 12% separating gels. The protein was electrophoretically transferred to a Nitrocellulose Transfer Membrane (Whatman GmbH, Dassel, Germany) and blocked with 5% BSA in TBS-T buffer (20 mM Tris base, 1.37 mM NaCl, 0.05% Tween-20, pH 7.6) overnight at 4°C. The membrane was incubated with anti-IκB-α antibody (Santa Cruz Biotechnolgy, Santa Cruz, CA, USA) and antiphospho-IκB-α antibody (Santa Cruz Biotechnology) for 2 hours. After washing with TBS-T three times, the membrane was incubated with HRP-conjugated secondary antibody in TBS-T at 1:2,000 dilution for 1 hour. The blots were processed with an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology) and were performed with Bio-Imagin RAS 1000 plus (Fugifilm, Tokyo, Japan).