Figure 1From: Sprouty is a cytoplasmic target of adenoviral E1A oncoproteins to regulate the receptor tyrosine kinase signalling pathwaySprouty proteins interact with E1A isoforms. (A) Schematic representation of the E1A wild type isoforms E1A13S, E1A12S, E1A9.5S, the deletion mutants ΔNE1A13S and ΔNE1A12S (with a deletion of the first 29 N-terminal amino acids) and Spry isoforms and deletion mutants (ΔNSpry1 with a deletion of amino acids 1-173; ΔCSpry1 with a deletion of amino acids 174-313). Red boxes in E1A proteins represent the conserved regions 1 to 4; the brown box in Spry represents the Spry domain and the blue box the conserved tyrosine phosphorylation site. (B, C) For GST pull-down assays HeLa cell extract, containing the isoform Spry1, Spry2 or Spry4 (B) or the Spry-mutants ΔNSpry1 or ΔCSpry1 (C) were incubated with the protein leader sequence GST- or GST-E1A-fusion proteins as indicated. 0.5 mg of cellular lysate and 40 μg of GST-E1A or GST were incubated at 4°C for 1 h. Proteins bound were subjected to SDS-PAGE, and immunoblot analysis was performed with antibodies directed against the Myc-epitope of Spry-fusion proteins. * The higher molecular weight band may represent post-translational modification of Spry1 (palmitoylation and phosphorylation) as reported before [22]. (D) For immunoprecipitation HeLa cells were transiently co-transfected with Spry1 and Myc-tagged E1A12S expression vectors as indicated. Cell extracts were prepared 48 h post transfection and incubated with anti-Spry1 antibodies. Interacting proteins were precipitated and analyzed on SDS-PAGE by Western blotting using anti-Spry1 and anti-Myc antibodies. Transient expression was examined in the total lysate (Δ co-eluted antibody heavy chains).Back to article page