Ab109749

Protein extracts (10 μg) prepared from LCLs or B lymphocytes were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Wisconsin, USA). The membranes were blocked with 5% milk in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and incubated overnight at 4°C with specific primary antibodies against the following proteins: TNF receptor-associated factor 1 (TRAF1, 1 : 1000, ab203316, abcam, Cambridge, UK), IκB kinase (IKK)-α (1 : 10000, ab109749, abcam), NF-κβ-inducing kinase (NIK, 1 : 500, ab203568, abcam), P52 (1 : 2000, ab129097, abcam), pro-caspase 3 (1 : 1000, ab32150, abcam), caspase 3 (1 : 500, ab49822, abcam), poly (ADP-ribose) polymerase (PARP, 1 : 2000, ab74290, abcam), and β-actin (1 : 5000, 66009-1-Ig, Proteintech, USA). After three washes with PBS/Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary goat antirabbit IgG (1 : 20000, PAB160011, Bioswamp) for 2 h at room temperature. Protein bands were visualized by enhanced chemiluminescence color detection (Tanon-5200, TANON, Shanghai, China) and analyzed using AlphaEase FC gel image analysis software.