Figure 1

DRB2 is found predominantly in the nucleus and forms a high molecular weight complex as well as a homo interaction. (a) Level of small RNA accumulation in wild-type (Col-0), drb2-1 and two complementing lines. Values are normalized to U6 RNA and are expressed as a ratio relative to Col-0. For p4-siRNAs, only the 24-nt species were used for normalization. (b) Subcellular localization of DRB2-GFP in a heterologous system. GFP signal is observed both in the cytoplasm and the nucleus, but is absent from the nucleolus. (c) Subcellular localization of DRB2-FlagHA by cell fractionation and western blot. DRB2-FlagHA appears to be mainly nuclear. Extracts from each compartment were loaded in a SDS-PAGE either as a fixed protein quantity (first three lanes) or as 1/100th of the total extract (last three lanes). C stands for cytoplasm, N for nucleus and P for pellet. DRB2-FlagHA is revealed with a commercial HA antibody (@), UGPase is used as the cytosol quality control and H3 as the nuclear quality control. (d) Coimmunopurification of the DRB2-Cmyc protein from DRB2-FlagHA bound Flag magnetic beads. DRB2-FlagHA is able to bind DRB2-Cmyc, while NERD-FlagHA is not. DRB2-FlagHA and NERD-FlagHA are both revealed with a commercial HA antibody and the presence of DRB2-Cmyc in the DRB2-FlagHA eluate is revealed with a Cmyc commercial antibody. (e) Gel filtration on a Superose 6 column of DRB2-FlagHA crude extracts. The elution profile of DRB2-FlagHA shows that it is present in a high molecular weight complex of an approximate mass of 2 MDa as well as in the intermediate forms of lower mass of this complex. Fractions (500 μl) were analysed by western blot, and DRB2-FlagHA is revealed with HA antibody. Fraction numbers, sizing standards and corresponding volumes are indicated.