20s proteasome α1

Endothelial cells were treated with prostaglandins for 4 h at 37°C. After incubation, the EC were solubilized in lysis buffer (1% Non-idet P-40, 0.1% SDS, 150 mM NaCl, 50 mM Tris–HCl, pH 7.2) and complete protease inhibitor (Roche Diagnostics, Germany). Immunoprecipitation of human plaques proteins was carried out by solubilizing the tissues in T-PER Tissue Protein Extraction Reagent (Thermo Scientific, IL, USA) and complete protease inhibitor (Roche Diagnostics, Germany) and subsequent homogenization in a tissue lyser. The samples were centrifuged (12,000 × g for 10 min at 4°C) and 300 μg of total cell extract was diluted to 500 μl with lysis buffer. The sample was incubated in 2 μg of antibody at 4°C for 2 h and 25 μl of Protein A/G plus agarose beads added to the sample prior to incubation overnight at 4°C. The immunoprecipitates were washed four times with lysis buffer and proteins were eluted with SDS sample buffer (63 mM Tris–HCl, 10% glycerol, 2% SDS, 0.0025% bromophenol blue, pH 6.8) and analyzed by SDS-PAGE, followed by Western blotting. Antibodies to PSMD2, PSMD3, PSMD11, 20S Proteasome α1, and 20S Proteasome β1 were obtained from Santa Cruz Biotechnology (CA, USA) and used for immunoprecipitation experiments.

NF-κB p65, p50, and p105 subunits antibodies and Ik-β and anti-rabbit secondary antibody were obtained from Cell Signaling Technology (MA, USA). The β-actin, anti-ubiquitin, anti-biotin, PSMD2, PSMD3, PSMD11, 20S Proteasome β1, 20S Proteasome α1, polyclonal anti-mouse secondary antibodies, and Protein A/G plus agarose beads were from Santa Cruz Biotechnology (CA, USA). Anti-PSMD1 and anti-TBP antibodies were from Sigma-Aldrich (Dorset, UK).