Figure 3.

Stability Regulation of ERF6 Protein in Vivo by Phosphorylation.

(A) and (B) Differential accumulation of ERF6^WT and phospho-mimicking ERF6^4D proteins in lines with similar levels of transgene expression. Twelve-day-old 35S:ERF6^WT and 35S:ERF6^4D transgenic seedlings (five independent lines each) were collected for protein and RNA preparations. Levels of ERF6 protein were determined by immunoblots using anti-myc antibody (A), and transgene expression at mRNA level was determined by real-time qPCR (B). Error bars indicate se (n = 3). Primers used for qPCR detect both endogenous ERF6 and transgene transcripts. Rubisco, ribulose-1,5-bis-phosphate carboxylase/oxygenase.

(C) Degradation of ERF6 protein by the proteasome pathway. Twelve-day-old 35S:ERF6^WT and 35S:ERF6^4D transgenic seedlings were treated with MG132, a 26S proteasome inhibitor, and samples were collected at different times. Levels of ERF6 protein were determined by immunoblots using anti-myc antibody. Equal loading of proteins was confirmed by Coomassie blue–stained gels (bottom panels).