Figure 5. FGF10- or GDNF-induced cellular spreading and proliferation mediated by LM-binding requires TRAF6-mediated K63-linked polyubiquitination of Akt.
(A-B) Itgα3f/fα6f/f and Itgα3−/−α6−/− CD cells were plated on LM-511 for 1 h, treated with FGF10 (A) or GDNF (B) (10 ng/ml each) and lysed at 5, 15 and 30 min later. Cell lysates were immunoprecipitated with an anti-Akt antibody and immunoblotted for K63-linked polyubiquitination or Akt. (C-J) Itgα3f/fα6f/f CD cells were transfected with non-silencing or TRAF6 siRNA (20 nM for 24 h), plated on LM-511 for 1 h and treated with FGF10 or GDNF (10 ng/ml each). (C-F) Cells were lysed 5, 15 and 30 min after addition of growth factors and immunoblotted for phosphorylation of Akt and TRAF6 protein levels (C-D) or immunoprecipitated with anti-Akt antibody and then immunoblotted for K63-linked polyubiquitination or Akt (E-F). β-actin was used as loading control (C-D). Cell spreading (G-H) and proliferation (J) were evaluated at 1 and 24 h after addition of growth factors, respectively. (G) Representative confocal images of the cells stained with rhodamine-phalloidin are shown; bar: 10 μM. (H) The individual measurements of cell surface (in pixels) of 15-30 cells with the mean is shown; *p≤0.05 between Itgα3f/fα6f/f CD cells transfected with non-silencing and TRAF6 siRNA. ≤0.05 between Itgα3f/fα6f/f untreated or treated with FGF10 or GDNF. (J) Proliferation as measured by the OD of BrdU-positive cells ±SEM of 4-6 independent experiments is shown. *p≤0.05 between cells transfected with nonsilencing and TRAF6 siRNA. ≤0.05 between Itgα3f/fα6f/f untreated or treated with FGF10 or GDNF.