Anti rpn7

NAC, Vit C, αLA, ATP, CBB-R250, EDTA, FeSO₄, glycerol, H₂O₂, imidazole, NaCl, NaH₂PO₄, NEM, Nonidet P40, PMSF, puromycin solution, SDS, Tris-HCl, Triton X-100, EZview^TM Red affinity gels (including streptavidin-HRP, anti-myc, and anti-FLAG^® M2), anti-actin (1:2000), and anti-FLAG M2 (1 μg/mL) antibodies were from Sigma-Aldrich (St. Luis, MO); USP2, anti-RPN7 (1:1000), and anti-ubiquitin (1:1000) antibodies from Enzo Life Sciences (Farmingdale, NY); anti-RPN6 antibody (1:1000) from Novus Biologicals (Littleton, CO); AcTEV protease, DTT, IPTG, MgCl₂, lipofectAMINE^®, PLUS^TM reagent, and anti-V5 (1:5000) antibodies from Life Technologies (Grand Island, NY); strepavidin-HRP conjugate (1:3000) from GE Healthcare (Piscataway, NJ); Bortezomib from LC Laboratories (Woburn, MA); anti-PCID2 (1:500) and anti-DSS1FL70 (1:500) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); complete EDTA-free protease inhibitor cocktail from Roche (Indianapolis, IN); anti-eIF3C antibody (1:15,000) from Bethyl Laboratories (Montgomery, TX); anti-His (1:500) antibody from EMD Biosciences (San Diego, CA); anti-RPN3 and anti-DSS1s3259-2 (1 μg/mL) antibodies from Proteintech (Chicago, IL); EcoRV, SgfI, and MluI restriction enzymes are from New England Biolabs (Ipswich, MA).

DNA fragments encoding proteasome subunits, chaperones, Rad23B and USP14 were amplified from corresponding cDNAs in the human ORFeome collection (v.5.1) and subcloned into pXF6F (3 × FLAG) vector (derived from pRK5, Genentech). HA-, MYC- and GST-tagged UBLCP1 were generated by PCR from UBLCP1 cDNA and subcloned into pXF4H vector (derived from pRK5, Genentech), pXF3HM vector (our laboratory) and pGEX-4T-1 (GE Healthcare), respectively. The deletions and point mutants of UBLCP1 were also generated by PCR. NLS-GFPu plasmid was kindly provided by Ron Kopito (Stanford University, CA, USA). The antibodies were purchased as follows: anti-α1–7 (#ST1049) and anti-phosphoserine (Phosphoserine Detection Kit, #525282) antibodies from EMD Millipore; anti-Rpn1 (#HPA045192), anti-FLAG (#F3165) and anti-γ-tubulin (#T5326) antibodies from Sigma; anti-UBLCP1 (#ab176340) and anti-histone H3 (#ab1791) antibodies from Abcam; anti-MYC (#sc-40), anti-GFP (#sc-8334) and anti-Cyclin D1 (sc-753) antibodies from Santa Cruz; anti-HA (#3724) and anti-p21^Cip1 (#2947) antibodies from Cell Signaling Technology; anti-Rpn2 (#PW 9270), anti-Rpn7 (#PW8225), anti-Rpt1 (#PW8825), anti-Rpt3 (#PW8175), anti-Rpt4 (#PW8220), anti-Rpt5 (#PW8770) and anti-Rpt6 (#PW8320) antibodies from ENZO Life Sciences; peroxidase-conjugated goat anti-rabbit and rabbit anti-mouse IgG (#315-035-048) from Jackson ImmunoResearch.