Pa28β

Specimens were fixed in 10% paraformaldehyde overnight, embedded in paraffin, serially sectioned (2.5 μM) and stained with hematoxylin eosin. Tissue sections were baked at 68°C for 2 h. Tissue sections were deparaffinized with 3 washes of xylene, followed by rehydration with successive washes of ethanol 100, 95, 90, 80, 70%. Samples then were stained with hematoxylin and eosin, successively dehydrated with washes of gradient alcohol and xylene dehydration, dried and mount before imaging. For IHC, blocked endogenous peroxidase by incubation in 3% H₂O₂ for 30 min and then washed three times per wash for 5 min in PBS. Sections were incubated with primary antibody at a dilution above overnight at −4°C, followed by incubation with rabbit antibody for 30 min at 37 °C. The enzyme was visualized after 3-min incubation with diaminobenzidine (DAB). Primary antibodies specific for CDK15 (GeneTex 1:50), PA28α (Cell signaling 1:100), PA28β (Cell signaling 1:100) were used for immunohistochemistry.

Bortezomib and carfilzomib were purchased from selleck chemicals. Other reagents were purchased from puromycin (Sigma), cycloheximide (Sigma), SUC-LLVY-AMC (Enzo Life Science), Z-Leu-Leu-Glu-AMC (Enzo Life Science), Boc-Leu-Arg-Arg-AMC (Enzo Life Science). Antibodies used were as follows: PA28α (Cell Signaling), PSMA2 (Cell Signaling), S5a (Cell signaling), PA28β (Cell Signaling), Phospho-eIF2α (Ser51) (Cell signaling), eIF2α (Cell signaling), α-tubulin (Genetex), PA28γ (Genetex), PSMB5 (Genetex), PSMB6 (Enzo life science), PSMB7 (Genetex), PSMB8 (Genetex), PSMB9 (R&D systems), PSMB10 (R&D systems), Rpt5 (Enzo life science), Ubiquitin (Cell Signaling), β-actin (Santa Cruz Technology), TCF11/NRF1 (Cell Signaling), LC3B (Cell Signaling), p62/SQSTM1 (MBL International) human Ig lambda light chain (R&D systems), actin (Sigma). pLKO.1 empty vector, shRNA vector targeting human PA28α, NRF1 siRNA (ON-TARGETplus SMARTpool), PA28α siRNA (Accell SMARTpool), and control siRNA were purchased from Dharmacon. Native Mark unstained protein standard was purchased from life technologies.

Cells were harvested and lysed, and protein concentrations were determined by BCA protein assay kit. Equal amounts of purified proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes. Western signals were detected using Enhanced Chemiluminescence kit (FDbio science). Primary antibodies were used as follows: CDK15 (1:300, GeneTex), PA28α (1:1,000, Cell signaling), PA28β (1:700, Cell signaling), GAPDH (1:1,000, Proteintech), β-Actin (1:1,000, Transkgen). Anti–rabbit or anti-mouse secondary antibody (Earthox) was used at 1:5,000 dilution.