Extended Data Fig. 3: FBXL7 binds c-SRC phosphorylated on Ser104.
a, Schematic representation of c-SRC mutants. Binding of c-SRC to FBXL7 is indicated with the symbol (+). b–d, HEK-293T cells were transfected with FLAG-tagged versions of either wild-type c-SRC or the indicated c-SRC mutants, or with an empty vector (EV). Twenty-four hours after transfection, whole-cell extracts (WCE) were subjected to immunoprecipitation (IP) with an anti-FLAG resin and immunoblotting. b–d, two independent experiments were performed with similar results. e, Lysates from HEK-293T cells were used in binding reactions with beads coupled to either a peptide or a phospho-peptide flanking the residue S104 in the c-SRC sequence (sequences shown on top of the panel). Bound proteins from three independent experiments were subjected to immunoblotting. f, Alignment of the region corresponding to amino acids 98–119 of human c-SRC in c-SRC orthologs. g, HEK-293T cells were transfected with FLAG-tagged FBXL7. Twenty-four hours after, whole-cell extracts were treated with λ-phosphatase for 4 h, then subjected to immunoprecipitation (IP) with an anti-FLAG resin and immunoblotting. h, Experiment was performed as in b–d. i, Surface structure of human c-SRC obtained from PDB [www.rcsb.org;57]. The amino acid S104 is highlighted in red. Its position shows that it is a solvent-exposed surface amino acid. j, HEK-293T cells were transfected with either one of two FLAG-tagged F-box proteins (FBPs), namely FBXL7 or FBXL1, or with an empty vector (EV). Twenty-four hours after transfection, whole-cell extracts (WCE) were subjected to immunoprecipitation (IP) with an anti-FLAG resin and immunoblotting. g, j: Two independent experiments were performed with similar results.
