Figure 6

IDEF1 undergoes proteasome-mediated degradation.

(a) Immunoblot analysis of IDEF1 protein in rice seedlings treated with MG132. The 14-day-old WT rice roots (R) and shoots (S) treated with DMSO (0 h) or 40 μM MG132 for 12 h and 20 h were used for protein extraction. Immunoblot analysis of native IDEF1 and modified IDEF1 (the high-molecular-weight, HMW) with an anti-IDEF1 antibody and HSP80 as loading controls. The arrow indicates unmodified IDEF1. (b) Immunoblot analysis of IDEF1 protein in rice suspension cells treated with 40 μM MG132 for 12 h and 20 h. (c) The histogram represents quantification of the IDEF1 and HMW-IDEF1 band in (b) using Image J. Protein levels are expressed as a ratio of IDEF1 or HMW-IDEF1 to HSP80. (d) Immunoprecipitation was performed using anti-IDEF1 antibodies on solubilised protein extracts from WT and subjected to immunoblotting with the anti-K48 polyUb antibody Apu2. IB, immunoblotting; IP, immunoprecipitation. The arrow indicates polyubiquitinated IDEF1. Equal protein loading was confirmed by CB (Coomassie Blue stain). Western blot using IDEF1 antibody was used as a loading control for the input of the IP. HC, heavy chain; HMW, high molecular weight. Error bars indicate SD (n = 3). Asterisks indicate P < 0.05 (*) and P < 0.01 (**) in Student’s t test analysis.