Fig. 5. SPA proteins physically interact with CO. (A) SPA proteins interact with CO in vitro. Recombinant ³⁵S-labelled CO was incubated with partially ³⁵S-labelled GAD-SPA proteins or GAD, respectively, and co-immunoprecipitated with anti-GAD antibodies. Supernatant fractions (1.6%) and 33.3% of the pellet fractions were resolved by SDS-PAGE and visualized by autoradiography using a phosphorimager. (B) Co-localization of CFP-SPA1 and YFP-CO in transiently transfected Arabidopsis leaf epidermal cells. Images were taken by confocal microscopy. Scale bar: 4 µm. (C) FRET microscopy by acceptor photobleaching. Fluorescence intensities of CFP-SPA1 and of YFP-CO or YFP constructs before and after acceptor photobleaching. Scale bar: 5 µm. (D) Comparison of FRET efficiency after acceptor photobleaching measured in nuclei. Data are mean±s.d. of 10-20 cells from three separate experiments.