Fig. 2.

OsHrd3 is required for polyubiquitination of unfolded proteins in rice endosperm. (A) The construct used for OsHrd3 knockdown (OsHrd3 KD); 35S P, Cauliflower mosaic virus 35S promoter (AF485783); HPT, hygromycin phosphotransferase coding region (K01193); Ag7 T, gene 7 terminator (AF85783); 16 kD P, promoter region of the gene encoding 16kDa prolamin (AY427574); RAPint, an intron from the rice aspartic protease gene (D32165); 16 kD T, 16kDa prolamin terminator. (B and C) Levels of polyubiquitinated proteins are reduced in OsHrd3 KD seeds. Seeds (14 DAF) from wild-type (WT) and OsHrd3 KD plants were dehulled and treated with either 0.1% dimethylsulphoxide (–) or 100 μM MG132 (+) for 24h and then treated with 20 μM PR-619 for 1h. Then, total proteins were extracted from the seeds with SDS–urea buffer containing 2-mercaptoethanol. The total proteins were separated by SDS–PAGE, followed by immunoblot analyses using an antibody against ubiquitin–protein conjugates (B) or Coomassie Brilliant Blue staining (C).