Figure 6: Nedd4 enhances IGF-I signalling in prostate cancer cells.

(a) The association of Nedd4 with IRS-2. DU145 cells or PC-3 cells were serum starved and cell lysates were subjected to immunoprecipitation and immunoblotting as indicated. (b,c) Effects of knockdown of Nedd4 or Epsin1 on IGF-I signalling in PC-3 cells. Cells transfected with indicated siRNAs were serum starved and then treated with or without 100 ng ml⁻¹ IGF-I for 1 min. Lysates were subjected to immunoprecipitation and immunoblotting as shown. Densitometric analyses of samples with IGF-I stimulation were performed. Tyrosine phosphorylation levels of IGF-IRβ and IRS-2 were normalized to their protein levels in immunoprecipitates. p85 PI3K bound to IRS-2 was normalized to IRS-2 levels in immunoprecipitates. Means±s.d. of three to five independent experiments are shown in the right panel. *Significant difference from control (P<0.05, one-way analysis of variance (ANOVA) followed by Tukey–Kramer test). (d) Effects of knockdown of IRS-2 or Nedd4 on IGF-I-induced DNA synthesis in PC-3 cells. Cells transfected with IRS-2 siRNAs or Nedd4 siRNAs were serum starved for 48 h and then treated with or without 100 ng ml⁻¹ IGF-I for 16 h. [³H]thymidine incorporation into DNA during 13–16 h of IGF-I treatment time was measured. The values were normalized and the means±s.d. of three independent experiments are shown. Right insert shows the immunoblotting results. *Significant difference (P<0.05, one-way ANOVA followed by Tukey–Kramer test).