DRB4 over-accumulates after MG132 treatment and in APC/C RNAi lines.
(A) Characterization of DRB4 overexpressing lines. Northern blot analysis (upper panels) of DRB4 mRNA accumulation in flower extracts from Col-0 and two independent transgenic lines expressing DRB4 under the control of the strong 35S promoter (referred hereafter as lines DRB4 OE-4 and -27). Accumulation of EF1α mRNA is used as a loading control. Western blot analysis (lower panel) performed using an antibody directed against DRB4. The asterisk indicates a non-specific cross-reacting band that can also be used as a loading control. Pictures of 5 week-old Col-0 and DRB4 OE-27 plants. (B) Three week-old DRB4 OE-27 seedlings were incubated in liquid medium supplemented or not with 100 µM MG132. After 4 h and 6 h of incubation, seedlings were collected and total protein extracted. Western blot was performed using antibodies against DRB4 or TSN as a loading control. (C) Western blot analysis of DRB4 accumulation in flower extracts from Col-0, drb4, dcl4, DRB4 OE-27 line, RNAi APC10-38 and RNAi APC6-20 lines. Coomassie staining was used as a loading control (LC).