Figure 6

Slit2N inhibition of VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs is Robo4-dependent. (A) PI3K activity by ELISA in L-LECs transfected with control siRNAs or Robo4-specific siRNAs, incubated with control or VEGF-C [100 ng/ml]; or after pretreatment with 10 nM Slit2N, then VEGF-C. Data represent the mean ± SD of 3 independent experiments (*p < 0.05; NS: not statistically significant). (B) Representative Western blot analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Total Akt used as loading control. (C) Quantitative analysis of Akt phosphorylation in L-LECs transiently transfected with control siRNAs or Robo4-specific siRNAs, and subsequently incubated with 10 nM Slit2N, VEGF-C [100 ng/ml]; or after pretreatment with Slit2N, then VEGF-C. Band intensity of each lane from Figure 6B was determined by densitometry. The ratio of p-Akt/total Akt in control siRNA-transfected L-LECs incubated with VEGF-C alone was set to “1” and all other ratios were determined vs. this control. Data represent the mean ± SD of 3 independent experiments (***p < 0.001).