Fig. 2. LFY and UFO physically interact. (A) UFO induces a supershift of a LFY-DNA complex. Electrophoretic mobility shift assay (EMSA) using an AP3 promoter sequence. BMV, non-specific brome mosaic virus control; LFY, in vitro transcribed and translated LFY protein; UFO, in vitro transcribed and translated UFO protein; LFY+UFO lane shows a supershift of the LFY-DNA complex, while UFO alone does not bind to the AP3 promoter sequence, indicating that UFO binds to the LFY-DNA complex. (B) Upper panel: GST pull-down assay showing interaction between a bacterially produced GST-LFY fusion protein and ³⁵S-labeled UFO protein; GST alone does not show an interaction with UFO. Input lane represents 10% of total protein. Lower panel shows same blot probed with GST antibody, demonstrating equivalent loading of GST and GST-LFY lanes. (C) Interaction of LFY and UFO in yeast two-hybrid assays. The AD-LFY/BD-UFO interaction shows a significant increase in β-gal activity over background controls. This interaction is enhanced 8.6-fold when the F-box is deleted in the AD-LFY/BD-FUFO combination compared with that of AD-LFY/BD-UFO. AD-FUFO and BD-LFY also shows a significant interaction, while AD-UFO and BD-LFY shows a background level of β-gal activity. AD, Gal4 activation domain; BD, Gal4 DNA-binding domain. Bars represent mean±s.e.m. for five replicates. (D) Co-immunoprecipitation of UFO-Myc with LFY-FLAG in planta. Protein from wild-type, 35S::LFY-FLAG, 35S::UFO-Myc; ufo-2 (LF; UM) or 35S::UFO-Myc (UM) floral buds were used to precipitate the LFY immune complex using -FLAG. For control immunoprecipitations, normal mouse IgG serum conjugated to agarose beads was used. Myc-tagged UFO specifically co-precipitated with LFY-FLAG from 35S::LFY-FLAG, 35S::UFO-Myc tissue only.