Anti p100 p52

To evaluate CD40-mediated activation we analyzed p100 processing and TRAF1 induction in U2OS cells and iDCs by western blotting. For western blot, coculture experiments were performed in six well plates (10⁶ +10⁶ cells per well). For stimulation of iDCs 0.8 × 10⁶ cells per well were seeded also in 6-well plates and stimulated overnight. Cells were collected in ice-cold PBS by scraping with a rubber policeman. Cells were then washed twice with fresh ice-cold PBS, pelleted (5 min, 4°C, 4630 g) and resuspended in Laemmli buffer. Samples were sonicated for 25 seconds at 100% amplitude with a sonication probe (UP100H Ultrasonic Processor, Helscher, Germany), heated at 95°C for 5 min and subjected to SDS-PAGE separation. After transfer of proteins to a nitrocellulose membrane western blot analysis was performed with an anti-p100/p52 (#05–361, Millipore), anti-TRAF1 (#4715), anti-A20 (#5630, both Cell Signaling Technology Beverly, MA, USA), anti-β-actin (#A1978–200), anti-Flag (M2) (#F-3165, both Sigma Aldrich) and horseradish peroxidase (HRP)-conjugated polyclonal rabbit anti-mouse antibody (#P0260, Dako, Glostrup, Denmark) or HRP-coupled anti-rabbit antibody (#7074, Cell Signaling Technology Beverly, MA, USA). Finally, membranes were developed by chemiluminescence western blot detection using ECL solution.