Fig. 3

c-SRC phosphorylates SMURF2 at Tyr314 and Tyr434. a Cell tracks of HGF-treated NBT-II cells at 24 h in presence or absence of the c-SRC inhibitor AZD0530 (1 μM). b Western blot analysis of NBT-II cells treated with 5 ng/ml of HGF and A83-01 (8 μM), or LY2157299 (1 μM), PD0325901 (1 μM), MEK162 (1 μM), AZD0530 (1 μM), or PP1 (1 μM). Lysates were collected at 90 min post HGF treatment and probed with indicated antibodies. c 293 T cells were transfected as indicated. Lysates were immunoprecipitated with anti-MYC. Whole cell extracts were probed with the indicated antibodies. d 293 T cells treated with HGF for indicated time points. Lysates were immunoprecipitated with anti-SMURF2. Whole cell extracts were probed with indicated antibodies. * indicates IgG band. e 293 T cells were transfected as indicated. Lysates were immunoprecipitated with anti-MYC affinity resin. Whole cell extracts were probed with the indicated antibodies. pY signifies tyrosine phosphorylation. f 293 T cells were transfected as indicated. After 24 h cells were treated with c-SRC inhibitors PP2 (1 μM) or dasatinib (1 μM). Twenty-four hours later, cells were lysed and immunoprecipitated with anti-MYC. Whole-cell extracts were probed with the indicated antibodies. g Schematic of c-SRC tyrosine phosphorylation sites on SMURF2. Red denotes identification by mass spectrometry. Green denotes identification through sequence alignment. h 293 T cells were transfected as indicated. After 48 h, cells were lysed and immunoprecipitated with anti-MYC. Whole-cell extracts were probed with the indicated antibodies. i 293 T cells were transfected as indicated. Whole-cell extracts were probed with the indicated antibodies. j 293 T cells were transfected as indicated. Whole-cell extracts were probed with the indicated antibodies. k 293 T cells were co-transfected with a CAGA-luciferase reporter and either SMURF2 WT, or SMURF2 (FF). Cells were stimulated with TGF-β overnight and luciferase activity was measured. l 293 T cells were co-transfected with a CAGA-luciferase reporter, SV40-Renilla and either SMURF2 WT, or SMURF2 Y314E, Y434E, or the double mutant EE. Cells were stimulated with TGFβ overnight and luciferase activity was measured. For k and l bars represent mean ± SD of three independent experiments. A two-tailed Student’s t test compares the treated populations, **P < 0.01, ***P < 0.001