hVAPB-mediated ER stress contributes to the accumulation of proteasome substrates.

Abstract

<p>(A) The immunoreactivity of CHOP in cells expressing hVAPB^WT and hVAPB^P56S was monitored by Western blotting (36 h after transfection). Thapsigargin treatment (for 16 h) was used as a positive control for ER stress-dependent CHOP upregulation. (B) Levels of BiP and phosphorylation status of IRE1 were examined by Western blotting 36 h following the transfection of cells with empty, hVAPB^WT and hVAPB^P56S plasmids. (C) Protein extracts of cells transfected with the proteasome reporters Ub-R-YFP and Ub^G76V-YFP and treated (or not) for 16 h with the ER stress inducer thapsigargin (10 µM) were subjected to Western blotting using anti-GFP (referred to as YFP), and anti-CHOP antibodies. (D) Quantification of the YFP immunoreactive bands (C) normalized to actin signals (arbitrary densitometry units). (E) Salubinal treatment (20 µm) diminished the accumulation of the proteasome reporter (YFP) as indicated by Western blotting of COS-7 cells co-transfectd with hVAPB^WT or hVAPB^P56S. (F) Differential detergent extraction and Western blot analysis of ubiquitin in cells expressing hVAPB^WT and hVAPB^P56S and treated or not with salubrinal (20 µM).</p