20s proteasome β1

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.

Monoclonal antibodies against human SIRT1, p53, 20S Proteasome β1, Mox1, gp91-phox, cyclin D1, N-cadherin and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and antibodies against vimentin, ubiquitin, acetyl-p53(Lys382), phospho-Rb(Ser807/811), Caspase-3, p21 Waf1/Cip1, Bcl-2, PARP, Akt, Bax and GAPDH were from Cell Signaling Technology (Danvers, MA, USA). The anti-E-cadherin antibody was purchased from BD Transduction Laboratories (Franklin Lakes, NJ, USA), and MG-132 (Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) was purchased from Calbiochem (Burlington, MA, USA). The anti-Nox4 antibody was purchased from Novus Biologicals (Littleton, CO, USA). The antibodies against proteasome subunit β type-2 were purchased from Abcam (Cambridge, UK), and proteasome subunit β type-5 was purchased from Proteintech (Rosemont, IL, USA). The proteasome 20S Activity Assay Kit and cisplatin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-mouse, anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. The primary antibody was diluted with 5% skimmed milk in 0.1%Tween20-TrisHCl-buffered saline (TBST) in the concentration range of 1:1000 to 1:2000; while the secondary antibodies were diluted in TBST solution in the concentration range from 1:10000 to 1:20000.

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).