Mcp231

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.

Antibodies and their sources were: anti-human ASPL (Abcam and Cell Signaling), anti-NSF (NSF-1 monoclonal purchased from Abcam), anti-his (Qiagen), anti-GFP (Invitrogen), anti-tubulin and anti-β-actin (Abcam), anti-human Rpn1 (p112, Enzo) and anti-20S proteasome α-subunits (MCP231, Enzo). The antibodies to p97 have been described previously [46 (link)]. Several antibodies were kindly provided by the following investigators: anti-calnexin (Prof. Ari Helenius, Zürich, Switzerland), anti-Pma1 (Prof. Per A. Pedersen, Copenhagen, Denmark), anti-CPY* (Prof. Jakob R. Winther, Copenhagen, Denmark).

The protein lysates of C. elegans or HEK293 cells were separated in an SDS-PAGE and transferred to a PVDF membrane (Trans-blot Turbo system, Bio-Rad). In this study the following antibodies were used: anti-α-tubulin (1:2,000, Sigma #T5168), anti-β-actin (1:2,000, Santa Cruz), anti-HSP-1/ HSP-110/DNJ-13 (1:5,000, (Scior et al., 2018 (link))), anti-DNAJB1 (1:1,000, Proteintech #13174-1-AP), anti-20S α-subunits (1:1,000, Enzo #MCP231), anti-LGG-1/SQST-1/p62 (1:1,000), anti-HSP-17 (1:2,500) and anti-LC3 (1:1,000, Abcam #ab48394), anti-mouse/rabbit HRP (1:10,000/1:15,000, Thermo Fisher #31444/#31460) or anti-mouse-Cy3 antibody (1:10,000, Licor #926-68072). LGG-1 and SQST-1/p62 antibodies were generated by immunizing rabbits with a synthetic peptide. LGG-1: PKSKLHDLDKKKYL; SQST-1/p62: AVPKPAQEPRIPPSPTSALPPPQFFN (Pineda, Germany). HSP-17 antibody was generated by immunizing rabbits with the purified protein (Pineda, Germany).