Anti β5

Equal amounts of protein extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Merk, Darmstadt, Germany). I-proteasome subunits were analyzed by immuno-blotting using anti-mouse anti-β5i/LMP7, anti-β5, anti-β1i/LMP2 (Abcam, Cambridge, UK) and anti-β1, anti-β2i/MECL-1, anti-α4 (self-made), and AP1 components, anti-(p)-cJun, anti-(p)-cFos, anti-cFos, anti-(p)-ATF-2, anti-ATF-2 (Cell signaling, Leiden, Netherlands), and anti-cJun (Santa Cruz Biotechnologie, Dallas, Texas, USA) antibodies combined with a secondary polyclonal mouse-anti-rabbit-IgG antibody conjugated to horseradish peroxidase (Dianova, Hamburg, Germany). Anti-β-actin antibody (Cell signaling, Leiden, Netherlands) was used as loading control.

BMDM and spleen specimen were lysed in extraction buffer containing (in mmol/L): Tris/HCl 50 (pH 7.4), KCl 154, glucose 5, EDTA 0.5, Complete protease inhibitor, and 1% Triton X-100. We isolated protein from livers and spleens separately for each animal, and used equal amounts of protein per animal to prepare one pooled sample per group. Total protein (10 μg per lane) was subjected to SDS-PAGE and membranes were probed with the respective antibodies: anti-Ubiquitin (DAKO), anti-β5 (Abcam), anti-β1 (Abcam), anti-β2 (Abcam), anti-β1i/LMP2 (Abcam), anti-β5i/LMP7 (Epitomics), anti-β2i/MECL-1, anti-α4 (kindly provided by the Department of Biochemistry, Charité-Universitätsmedizin Berlin, Germany). After probing with the respective secondary antibodies, bands were visualized using ECL detection system (Amersham) and chemiluminescence system Fusion Solo (Vilber Lourmat). Amidoblack staining or probing with antibodies against GAPDH (Santa Cruz) or β-actin (MAB150, Millipore) served as controls for equal protein loading. Densitometry was performed using ImageJ software.