Figure 5

Slit2N inhibits VEGF-C-induced activation of VEGFR-3 in 293/VEGFR-3 transfectants and L-LECs in a Robo4-dependent manner. (A) Representative Western blot analysis of Robo1 and Robo4 expression in L-LECs, HMVECs, and 293/VEGFR-3 cells. GAPDH used as loading control. (B) Representative Western blot analysis of Robo4 and Robo1 expression in 293/VEGFR-3 cells, 24 h after transfection with pCMV-RFP (vector) or pCMV-RFP-Robo4 (Robo4). GAPDH used as loading control. (C) Representative VEGFR-3 IP/Western blot analysis of phosphorylated VEGFR-3. 293/VEGFR-3 cells were transfected with pCMV-RFP (vector) or pCMV-RFP-Robo4 (Robo4). After 48 h, cells were incubated with a control (“- -”), 10 nM Slit2N, or VEGF-C [100 ng/ml]; or pretreated with Slit2N, then VEGF-C. Total VEGFR-3 used as loading control. (D) Quantitative analysis by densitometry of Figure 5C. The ratio of p-VEGFR-3/VEGFR-3 in vector-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). (E) Representative Western blot analysis of Robo4 and Robo1 expression in L-LECs, 24 h after transfection with control siRNAs or Robo4-specific siRNAs. GAPDH used as loading control. (F) Representative VEGFR-3 IP/Western blot analysis of phosphorylated VEGFR-3 in L-LECs transfected with control siRNAs or Robo4-specific siRNAs, after incubation with control, VEGF-C [100 ng/ml]; or pretreatment with 10 nM Slit2N, then VEGF-C. Total VEGFR-3 used as loading control. (G) Quantitative analysis by densitometry of Figure 5F. The ratio of p-VEGFR-3/VEGFR-3 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 < 0.05).