Enhanced chemiluminescence ecl detection system

Each cytosolic and nuclear sample was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (Cytiva, Marlborough, MA, USA), which were blocked in BSA for 1 h. The blots were incubated with antibodies against phospho-IκBα (p-IκBα), GFAP, and GAPDH (Santa Cruz Biotechnology, Inc.); p65 (Cell Signaling Technology); and phospho-p65 (p-p65; Invitrogen) in blocking solution overnight at 4 °C. The membranes were washed with PBST and then incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. The membranes were developed using the Enhanced Chemiluminescence (ECL) detection system (Santa Cruz Biotechnology, Inc.). Band densities were quantified using FUJIFILM Science Lab Image Gauge Ver. 4.0 (Fuji Photo Film Co., Ltd., Tokyo, Japan).

Total proteins from uninfected and 263K-infected plasma (terminal stage) were subjected to electrophoreses on 10% polyacrylamide gel and blotting onto nitrocellulose membrane as described previously [10 (link)]. The membranes were blocked in TBS-T for 1 h at room temperature and then incubated with biotinylated-lectins (ConA, WGA, RCA, DBA, PNA, SBA, UEA-1) diluted in TBS-T at a final concentration of 1 μg/mL for 2 h at room temperature. After washing six times with TBS-T, membranes were incubated with a streptavidin HRP-conjugated diluted in TBS-T for 1 h at room temperature. Signals were visualized on X-ray film (Amersham) by using the Enhanced Chemiluminescence (ECL) detection system (SantaCruz).