Figure 5

SPL7 homodimerizes in vivo . (a) For a pull-down assay by means of an antibody against HA (BOUND), total protein was extracted from tobacco leaves transiently expressing full-size SPL7 tagged either with GFP or HA epitopes (GFP::SPL7 and HA::SPL7; INPUT). Input and bound fractions were assayed by Western blot using an anti-GFP antibody to assess GFP::SPL7 co-immunoprecipitation. Membranes were reprobed with an anti-HA-HRP (anti-HA) antibody to check for HA::SPL7 pull-down. Sizes of molecular-weight markers run on the same gels are indicated at the left according to manufacturer's indications for precasted gels. Note that the apparant molecular weights may differ in comparison to those shown in Figure 2 due to the use of a different separation matrix for electrophoresis. (b) For bimolecular fluorescent complementation (BiFC) analysis the split-YFP tags were N-terminal fused to the full-size SPL7 protein (nYFP::SPL7, cYFP::SPL7) and co-expressed in tobacco leaves. Restoration of the YFP fluorescence signal could be observed with widefield epifluorescence microscopy using a YFP band-pass filter. Co-expression of the individual constructs with their complementary empty vectors (middle and right panels) did not result in reconstitution of YFP fluorescence. (c) A representative confocal microscopic image of the reconstituted YFP fluorescence illustrating its predominant extranuclear localization is shown together with chlorophyll autofluorescence and merged images. Scale bars, 25 μm.