Figure 3.

In Vitro Ubiquitination Assays Showing the Degradation of OsELF3 by HAF1.

(A) to (C) HAF1 acts as an E3 ligase to mediate the ubiquitination of OsELF3. GST-OsELF3 ubiquitination assays were performed using MBP-HAF1 (or MBP) as a negative control (A). Ubiquitinated GST-OsELF3 was detected by His antibody (B) and OsELF3 antibody (C). Molecular masses of ubiquitinated GST-OsELF3 in kilodaltons are indicated on the right.

(D) to (F) HAF1 mediates the degradation of OsELF3 in vitro. Ten micrograms of total protein extracts was loaded into each lane, and ACTIN was used as a loading control. MBP was used as a negative control. MG132 is a proteasome inhibitor.