Pr 957

The total proteins were extracted and separated by 10% or 15% SDS-PAGE and were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, Massachusetts, USA) using a semi-dry Gel Transfer Device (Bio-Rad, Hercules, California, USA). The membranes were blocked using 5% non-fat milk and probed with primary antibodies and HRP-conjugated secondary antibodies. Antigen-antibody complexes were visualised using a chemiluminescent ECL detection system and analysed using a ChemDoc XRS+image analyser. GAPDH or β-actin was used as an internal control. The antibodies used included anti-β5i (1:5000, ab180606, Abcam), anti-RIG-I (1:2000,3743, Cell Signaling Technology), anti-Phospho-NF-κB p65 (1:1000, 3033, Cell Signaling Technology), anti-Phospho-IRF3 (1:500, ab76493, Abcam), anti- IFNβ (1:500, ab275880, Abcam), anti- MxA (1:500, sc-166412, Santa Cruz), anti-MuRF1 (1:2000, ab172479, Abcam), anti-β-actin (1:5000, YM3028, Immunoway) and anti-GAPDH (1:10000, YM3029, Immunoway). The selective β5i inhibitor PR-957 was purchased from Selleck Chemicals (Houston, Texas, USA).

For PR957 treatment, HSMMs were seeded into six-well plates (Nunc, Roskilde, Denmark). Next, PR957 (Selleck, Houston, Texas, USA), at a concentration of 100 nM was added to the culture medium.

Corilagin analytical standard (purity, >99%) was purchased from Sigma-Aldrich (Merck KGaA) and TMZ from Selleck Chemicals. Anti-β1, anti-β2, anti-β5, anti-β1i, anti-β2i and anti-β5i antibodies were procured from Abcam; anti-β-actin and horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit IgG secondary antibodies were acquired from OriGene Technologies, Inc.; MG-132, bortezomib, carfizomib and PR-957 were procured from Selleck Chemicals. DMEM, streptomycin penicillin, and fetal calf serum were obtained from HyClone (GE Healthcare Life Sciences); trypsin was obtained from Gibco (Thermo Fisher Scientific, Inc.). BCA protein assay kit was purchased from Beyotime Institute of Biotechnology, an Annexin V/PI apoptosis detection kit from BD Biosciences and proteasome activity detection kit from Promega Corporation.