Psma2

1DE and 2DE protein immunoblots (IB) were conducted as previously described [14 (link)]. Total cell lysate samples were separated by 2DE (n = 3 HG, n = 3  NG^∗). For 2DE IB analysis, following IEF of mesangial proteins, the plastic backing of the IPG strips was trimmed off. The acidic most point of the strip was aligned in the IPG well of the Bis-Tris mini gels adjacent to the MW standard lane of the minigel. This procedure insured uniform alignment of IPG strips to the MW standards, in order to compare PHB migration pattern between experimental conditions. Following 1DE or 2DE electrophoresis and transfer, membranes were immunoblotted for PHB (Santa Cruz Biotechnologies, Santa Cruz, CA) at a 1 : 1000 dilution in 5% albumin in Tris-Tween-20 buffered saline (TTBS). PHB spots were imaged on film with luminol images aligned and quantified by densitometry analysis comparing the means of the acidic third and basic third of the PHB charge trains to the total train densitometry. Additional antibodies used for 1DE immunoblots were anti PDI (Stressgen; San Diego, CA) at a concentration of 1 : 10,000 and PSMA2 (Cell Signaling; Danvers, MA) at a concentration of 1 : 1000.

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.