Fig. 6.

OsHrd3 is required for polyubiquitination of RM1. Seeds (14 DAF) from wild-type (WT) and OsHrd3 KD plants were dehulled and treated with 100 μM MG132 for 24h, and then with 20 μM PR-619 for 1h. (A) Total proteins were extracted with SDS–urea buffer supplemented with 2-mercaptoethanol and separated by SDS–PAGE, followed by immunoblot analyses using antibodies against RM1. Total proteins were extracted from the seeds and then immunoprecipitated with antibody against ubiquitin–protein conjugates. Total proteins (2%) and immunoprecipitated proteins (IP) were separated by SDS–PAGE, followed by immunoblot analyses using antibodies against the ubiquitin–protein conjugates (B) and RM1 (C). W and H represent samples from WT and OsHrd3 KD seeds, respectively.