Western Blot Analysis of CD83 Expression

Whole cell extracts for SDS-PAGE were generated as follows (26 (link)): 1 × 10⁶ BMDCs or B cells were washed with ice-cold PBS and resuspended in 50 μl lysis buffer mixed with Na₃VO₄, NaF, and PMSF. Afterwards, protein concentrations were determined by the BCA protein assay described above. Sample proteins were boiled in the presence of a loading dye mixed with SDS and 2-ME for 10 min at 95°C. Then, 20 μg of total protein per sample were separated on a 12.5% polyacrylamide gel and afterwards transferred onto nitrocellulose filters (Schleicher & Schüll/GE Healthcare), with a pore size of 0.2 μm with the wet blotting device “Mini-Protean II Cell and System” (BioRad). Membranes were incubated with primary antibodies against human CD83 (1G11, kindly provided by Elisabeth Kremmer, LMU Munich, Germany), murine CD83 (R&D Systems) or Beta-actin (AC-74; Sigma-Aldrich), followed by HRP-conjugated goat anti-rat IgG (Cell Signaling Technology), donkey anti-goat IgG (Promega), or goat anti-mouse IgG (Promega) antibodies. Detection was performed with the chemiluminescent ECL Prime Western Blotting Detection Reagent (Amersham, GE Healthcare) on a high performance chemiluminescence film (GE Healthcare).