Ecl western blotting detection reagents and analysis system

For analysis of NIK accumulation, p100 to p52 processing, expression of TRAF1 and TWEAK, and phosphorylation and degradation of IκBα cells were washed once with PBS, harvested using a rubber policeman, centrifuged, and then directly lysed in 4× Laemmli sample buffer (approximately 1 × 10⁶ cells per 100 μl buffer; 5 min, 95°C) supplemented with complete protease inhibitor from Roche Applied Science and phosphatase inhibitor mixtures I and II from Sigma. Lysates were sonicated for 15 s with maximum amplitude (UP100H Ultrasonic Processor, Hielscher, Germany), heated for 5 min at 95°C and centrifuged for 3 min (Eppifuge, full speed) to remove residual insoluble debris. Lysates were further processed using standard protocols for SDS-PAGE and immunoblotting using horse radish peroxidase-conjugated secondary antibodies (Dako) and the ECL Western blotting detection reagents and analysis system (Amersham). Primary antibodies used were anti-IκBα (clone C35A5, Cell Signaling), anti-pIκBα (clone 14D4, Cell Signaling), anti-NIK (#4994, Cell Signaling), anti-p100/p52 (#05-361, Upstate), anti-TRAF1 (H-132, Santa Cruz), anti-TWEAK (#AF1090, R&D Systems,) and anti-tubulin (clone DM1A, Neomarker).