Fig. 3.
SERCA2 mutants are polyubiquitinylated and form aggregates in the aggresome. (A,B) Mutants were not degraded by proteasomes. HEK293 cells were transfected with FLAG-tagged expression plasmids of WT or mutant SERCA2 for 24 hours before treatment or no treatment with 2.5 μM MG132 for an additional 16 hours. Anti-FLAG antibody was used to detect the expression of WT and mutant proteins by western blotting (A) or immunostaining (B). (C) Polyubiquitinylation of SERCA2 mutants. After 36 hours of transfection with FLAG-tagged WT or mutant SERCA2 expression plasmids, the cells were lysed with 1% Triton X-100 and supernatants were immunoprecipitated by anti-FLAG antibody. Anti-ubiquitin blotting detected polyubiquitinylated proteins in immunoprecipitates. Densitometric quantification of respective polyubiquitin relative to total protein of WT or mutant SERCA2 is shown in the bottom of the panel. (D) Mutant proteins form aggregates in aggresomes. HEK293 cells were transfected with 0.1 μg/ml FLAG-tagged WT or mutant SERCA2 expression plasmids for 24 hours. Then the transfected cells were fixed and double stained with antibodies to mouse FLAG and to each of the following proteins: rabbit ubiquitin (Ub), 20S proteasome, or goat vimentin. Representative results from mutant A838P are shown.