Figure 4

Slit2N inhibits VEGF-C-enhanced PI3K activity and Akt phosphorylation in L-LECs. (A) Representative Western blot analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Total ERK1/2 used as loading control. (B) Quantitative analysis of phosphorylated ERK1/2 in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4A was determined by densitometry. The ratio of p-ERK1/2 to total ERK1/2 of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments. (C) PI3K activity by ELISA in L-LECs incubated with various concentrations of Slit2N and/or VEGF-C [100 ng/ml]. Data represent the mean ± SD of 3 independent experiments (**p < 0.01, ***p < 0.001). (D) Representative Western blot analysis of phosphorylated Akt in L-LECs incubated with a control, VEGF-C [100 ng/ml]; or preincubated with various concentrations of Slit2N, then VEGF-C. Total Akt used as loading control. (E) Quantitative analysis of phosphorylated Akt in L-LECs, after treatment with control, VEGF-C alone [100 ng/ml]; or after preincubation with various concentrations of Slit2N, then VEGF-C. Band intensity of each lane from Figure 4D was determined by densitometry. The ratio of p-Akt to total Akt of L-LECs incubated with VEGF-C alone was set to “1” and values of all other ratios were calculated vs. this control. Data represent the mean ± SD of 3 independent experiments (**p < 0.01).