Fig. 5.

ER-targeted products of stalled ribosomes are degraded by the proteasome. (a) Western blots of extracts and supernatants of transfected cells treated with cycloheximide (CHX) for the indicated time periods. Representative of n = 2 independent experiments. (b) Western blots of extracts of cells treated with CQ or MG132 (MG) for 4 h. Representative of n = 3 independent experiments. (c) Anti-HA Western blot of anti-SV5 immunoprecipitates of cells co-transfected with the indicated constructs and a HA-tagged ubiquitin encoding plasmid. When indicated, (+) cells were treated with MG132 for 4 h. Representative of n = 2 independent experiments. (d–e) Western blots of extracts of cells co-transfected where indicated (+) with OTU, p97QQ or YOD1-C160S (C160S) encoding plasmids. Representative of n = 3 independent experiments. (f) Western blot of cells transfected with plasmid encoding the 2A-P* or 2A* MHC-Iα membrane protein and treated with MG132 for 4 h or co-transfected with OTU, as indicated. Representative of n = 3 independent experiments. (g) Upper panels: Western blot of extracts of cells transfected with siRNA for Listerin (Ltn) or an irrelevant siRNA (irr) and 48 h later co-transfected with the indicated constructs and EGFP as control. Lower panel: ethidium bromide stained agarose gel showing RT-PCR amplification of a 290-bp fragment of Ltn mRNA from cells transfected with the irrelevant or the Listerin-specific siRNA. Representative of n = 2 independent experiments.