Cd154

Protein extracts were precleared by incubating with protein G agarose beads for 2 h. The beads were removed by centrifuging the samples at 5,000 g for 5 min. Supernatant was collected, a portion of the supernatant was collected and used as the input; the supernatant was incubated with antibodies [CD83 or CD154 (Santa Cruz Biotech); final concentration: 1:100] or isotype IgG overnight. Immunocomplexes in the samples were precipitated by incubating with protein G agarose beads for 2 h. The beads were collected by centrifugation. Proteins on the beads were eluted with an eluting buffer and analyzed by Western blotting. All the procedures were performed at 4 °C. (Reagents and materials for IP were purchased from Invitrogen).

Cells were collected from relevant experiments. In surface staining, cells were stained with fluorescence-labeled antibodies [CD3 (Alexa Fluor® 488), CD4 (Alexa Fluor® 546), IL-4 (Alexa Fluor® 594), IL-5 (Alexa Fluor® 647), IL-13 (Alexa Fluor® 680), CD154 (Alexa Fluor® 790), CD83 (FITC); Santa Cruz Biotech], [Bcl2L12 (APC); Abcam] (diluted at 1:100) or isotype IgG for 30 min on ice. In intracellular staining, cells were fixed with paraformaldehyde (1%; containing 0.1% Triton X-100) for 1 h and then stained with fluorescence-labeled antibodies of interest or isotype IgG for 30 min on ice. After washing with phosphate buffered saline (PBS) thoroughly, cells (10⁶ cells/sample) were analyzed with a flow cytometer (FACSCanto II). Data were analyzed with a software package flowjo (TreeStar Inc., Asland, OR) with the data obtained from isotype IgG staining as gating references.