Figure 4

Over expressing FBW2 decreases ARGONAUTE1 (AGO1) protein levels. (A) Primary transformants containing a genomic 35S::FBW2 (FBW2ox) transgene. The percentage of plants displaying each phenotype is indicated (n = 345 plants). (B) L1 plants with, and without, the FBW2ox construct. FBW2ox restores the expression of the 35S::GUS transgene present in L1. (C) Western blot of protein extracts from 14-day-old seedlings probed with an anti-AGO1 antibody. AGO1 is reduced in plants containing FBW2ox. Actin was used as a loading control. (D) The proteasome inhibitor MG132 has no effect on AGO1 protein levels. Western blots of protein isolated from plants treated as indicated were probed with anti-AGO1 antibody. The lower anti-AGO1 blot is a longer exposure. The increased abundance of ubiquitinated proteins in MG132-treated plants indicates that the treatment was effective. Ponceau staining and a nonspecific band (*) were used as a loading control. (E) Abundance of miRNA targets in various genotypes as measured by quantitative-real time polymerase chain reaction. The abundance of these transcripts is significantly greater in FBW2ox sqn-1 than in the parental lines, suggesting that sqn-1 and FBW2ox operate independently to reduce AGO1 activity. Results were normalized to EIF4 (± standard deviation).