Immunoprecipitations
Immunoprecipitation of CD40 was performed by activated receptor capture, as previously described [6]. Briefly, 3×107 cells (A20.2J and derivatives) were incubated for 60 minutes at room temperature with 10 µl magnetic protein G beads (Dynal) pre-coated with 10 µg goat anti-rat IgG (Jackson), and 10 µg anti-CD40 (1C10) or an isotype control antibody (mAb72). Cells/beads were then pelleted by centrifugation and lysed in buffer containing 1% Triton X100. Beads were washed with lysis buffer to remove unbound material. In some experiments, material associated with the beads was dephosphorylated with lambda phosphatase (New England BioLabs) as per manufacturer's instructions. Beads were resuspended in 2X SDS-PAGE sample buffer and heated for 5 minutes at 95°C. Material eluted from the beads was fractionated by SDS-PAGE and transferred to PVDF membranes for Western blotting. In some experiments, cells were cultured for 6 hrs with 25 µM antennapedia-linked SMAC-N7 peptide (Calbiochem) or an appropriate volume of the solvent used for the peptide (DMSO). After incubation, cells (in peptide- or DMSO-containing medium) were stimulated with antibody-coated beads as outlined above.