P62 sqstm1

Briefly, liver sections (4 µm thick) were subjected to antigen retrieval and block of endogenous peroxidase activity and avidin-biotin-peroxidase method according to previous study [40 (link)]. The slides were incubated with normal serum from species producing the secondary antibody and subsequently with primary polyclonal antibodies against 4HNE (1:400, Abcam, #ab46545, Cambridge, UK), SIRT1 (1:150, Santa Cruz Biotechnology, #sc15404, Dallas, TX, USA), GRP78 (1:300, Abcam, #ab21685), SREBP1 (1:100, Santa Cruz Biotechnology, #sc8984), IL6 (1:100, Santa Cruz Biotechnology, #sc1265), p62/SQSTM1 (1:50, MBL International, Woburn, MA, USA) or monoclonal antibodies against F4/80 (1:50, Bio Rad, #MCA497GA, Segrate, Italy), Mitofusin 2 (1:200, Abnova, #H00009927, Taipei, Taiwan). All experiments were performed in triplicate. The staining intensity was expressed as arbitrary units (AU) or percentage of positive nuclei, in 20 randomly chosen microscopic fields, using an image analyzer (Image Pro Premier 9.1, Media Cybernetics, Rockville, MD, USA).

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.

Protein samples from whole cell lysates were analyzed by SDS-PAGE and Western blot, as described previously [18] (link) Blots were probed with primary antibodies specific for: Atg3 (Abcam), Atg5 (Novus Biologicals), p62/SQSTM1 (MBL international), LC3 (MBL international), Gbp2, β-actin (Sigma). Binding of secondary HRP-labeled goat-anti-rabbit or goat-anti-mouse antibodies (Thermo Scientific) was analyzed using SuperSignal^(R) West Pico or West Femto Chemiluminescent Substrate (Thermo Scientific).