Formation of a Fuss/Med protein complex and its translocation into the nucleus.

(A) Yeast Two Hybrid experiment showing physical interaction of Fuss and Med: pPC97-Fos + pPC86-Jun: interaction control; pCL1+ pPC86: Gal4- growth control and empty prey vector; pdbLeu-Fuss + pPC86: negative control; pdbLeu-Fuss + pPC86-Med: positive interaction of Fuss and Med. (B) Coimmunoprecipitations: in lysates from S2-cells co-expressing HA-Fuss together with FLAG-Med or FLAG-Mad, HA-Fuss co-precipitates with FLAG-Med but not with FLAG-Mad. In lysates from S2-cells co-expressing FLAG-Fuss with HA-Med or HA-Mad, HA-Med co-precipitates with FLAG-Fuss and HA-Mad does not. (C) Wing of a male fly of the genotype A9-Gal4; UAS-fuss-GFP. Overexpression of fuss-GFP leads to truncations of L2 and L5 veins and a loss of the p-cv. (D–E”) Upon co-expression of Med, Fuss-GFP is partially translocated into the nucleus while the cytoplasmic fraction of the protein is reduced. Confocal scans of L3 wing discs stained with anti-GFP (D, E) and anti-Histone (D’, E’) antibodies. (D–D”) A9-Gal4; UAS-fuss-GFP. The Fuss-GFP fusion protein is mainly localised in the cytoplasm. (E) A9-Gal4; UAS-fuss-GFP; UAS-Med. Arrows emphasize some of the cells that clearly show nuclear localisation of the Fuss-GFP fusion protein. (F–G”) Fuss does not inhibit the nuclear translocation of pMad. Confocal scans of L3 wing discs stained with anti-phospho-SMAD1/5 (F, G) and anti-Histone (F’, G’) antibodies. (F–F”) A9-Gal4. (G–G”) A9-Gal4; UAS-fussB. All Scale bars: 10 µm.