Fig. 4

c-SRC phosphorylation of SMURF2 inhibits SMAD7 binding. a 293 T cells transfected with MYC-SMURF2, Flag-SRC, control vector and HA-tagged ubiquitin. Following immunoprecipitation of MYC-SMURF2 lysates were resolved by SDS–PAGE and probed with indicated antibodies. b, c Representative structure from MD simulation of WW3 domain of SMURF2 in complex with a peptide from SMAD7 in b unphosphorylated state of Y314 and c phosphorylated state of Y314. The WW3 domain is shown in electrostatic surface and ribbon representation, whereas the SMAD7 peptide is shown in yellow colour in ribbon. The PY motif of SMAD7 and Y314 along with the adjacent arginine residues of WW3 domain is shown in stick representation. d Time series plot (in angstrom unit) of the centre of mass distance between Y314 of WW3 domain and P209 from SMAD7 peptide in the phosphorylated (red colour) and unphosphorylated (black) states of Y314 during the MD simulation of WW3-SMAD7 complex. e 293 T cells were transfected as indicated with MYC-tagged SMURF2 or corresponding mutants and FLAG-tagged SMAD7. After 48 h, cells were lysed and immunoprecipitated with anti-MYC affinity resin. Whole-cell extracts were probed with the indicated antibodies. f 293 T cells were transfected as indicated with MYC-tagged SMURF2, Flag-tagged SMAD7 and/or Flag-tagged c-SRC. After 48 h, cells were lysed and immunoprecipitated with anti-MYC affinity resin. Whole-cell extracts were probed with the indicated antibodies. g 293 T cells were transfected as indicated with MYC-tagged SMURF2, MYC-tagged SMURF2(C/A) or MYC-tagged SMURF2(FF), Flag-tagged SMAD7 and/or Flag-tagged c-SRC. After 48 h, cells were lysed and immunoprecipitated with anti-MYC affinity resin. Whole-cell extracts were probed with the indicated antibodies