Proteasome Subunit Immunodetection Assay

Anti-20S core α subunits monoclonal mouse antibody (mAb MCP231) and Rpt6 regulatory subunit monoclonal mouse antibody (mAb p45-110) were purchased from Enzo Life Sciences. Rabbit PSD-95 antibody was purchased from Calbiochem. Custom rabbit (pAb; clone #07) anti-Rpt6 phospho-specific antibody for S120 (pS120) was previously generated commercially (ProSci) against a synthetic phosphorylated peptide (Djakovic et al., 2012 (link)). Fluorescent and HRP-conjugated secondary antibodies were purchased from Thermo Fisher Scientific. N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) substrate was used (BACHEM). GST-Ubl and His-UIM constructs were a gift from the Alfred Goldberg lab. Malachite Green and Adenosine triphosphate were purchased from Sigma-Aldrich. PhosSTOP kinase/phosphatase inhibitor and cOmplete protease inhibitors were purchased from Roche. Picrotoxin was purchased from Tocris.