2.2.7. p100 to p52 Processing and Western Blotting Analysis

HT1080 cells (1 × 10⁶/well) were seeded in 6-well plates and cultivated overnight. Cells were then treated with AuNPs, anti-Fn14 antibodies, and AuNPs-immobilized anti-Fn14 antibodies. As a positive control, cells were also stimulated with Flag-TWEAK [25]. The next day, cells were collected using a rubber policeman and the remaining medium was removed via two washes with ice-cold phosphate buffered saline (PBS). Total cell lysates were then prepared in 4× Laemmli sample buffer (8% SDS, 0.1 m DTT, 40% glycerol, 0.2 m Tris (pH 6.8), 0.004% bromophenol blue) supplemented with complete protease inhibitor (Roche Applied Science, Grenzach-Wyhle, Germany) and phosphatase inhibitor mixtures I and II (Sigma-Aldrich, Schnelldorf, Germany) via sonication (10 pulses for 20 s) and boiling (95 °C, 5 min). Lysates were cleared via centrifugation (14,000 rpm, 4 °C, 10 min) and separated using sodium dodecylsulfate polyacrylamide gel electrophoresis on 12.5% gels. After transfer to nitrocellulose TRAF1 induction, TRAF2 degradation and p100 to p52 processing were analyzed using western blotting. In brief, free protein binding sites on the nitrocellulose were blocked for 1 h in Tris-buffered saline containing 0.1% Tween 20 and 5% dry milk and proteins of interest were detected via sequential incubation with primary antibodies: (anti-TRAF1 mAb, (45D3) #4715, Source: Rabbit; anti-TRAF2 mAb, Source: Rabbit, (C192), #4724) from Cell Signaling, MA, USA; anti-NFκB p100/p52 mAb, Source: Mouse, #05-361 from Millipore, Darmstadt, Germany; anti-ß-actin mAb, Source: Mouse, clone AC-15, #A1978 from Sigma-Aldrich, Schnelldorf, Germany, horseradish peroxidase(HRP)-conjugated secondary antibodies: (Anti-Mouse Immunoglobulins/HRP, Source: Rabbit, #P0260 from Dako, Hamburg, Germany; Anti-rabbit IgG, HRP-linked Antibody, Source: Goat, #7074, Cell Signaling, MA, USA), and the commercially available ECL western blotting detection reagents and analysis system (Amersham Biosciences, Muenchen, Germany).