Fig. 6.

Stalling of membrane-bound ribosomes triggers ERAD. (a) Western blot-retardation assay (WB-ra) of extracts of cells co-transfected with BAP-tagged constructs terminating in 2A-P* or 2A* and a cytosolic biotin ligase BirA encoding plasmid. Migration of in vivo biotinylated proteins is retarded in the presence (+) of StrAv. (b) WB-ra as in panel a, in extracts of cells treated with MG132 or co-transfected with OTU or p97QQ, as indicated. (c) WB-ra of extracts of cells co-transfected, where indicated, with the BiP/GRP78 mutant T37G (BiP T37G) encoding plasmid. (d, Scheme of constructs with the N-glycosylation tag (N-2A-P* and N-2A*, left panel) and Western blot upon co-transfection with the BiP T37G encoding plasmid. Arrowheads indicate the glycosylated and de-glycosylated products. All panels are representative of at least three independent experiments.