Figure 4
Figure 4. VEGF-A–induced phosphorylation of Syk (Y323). (A) HDMECs were serum-starved overnight in media containing 2% FBS. Cells were washed and incubated with either 0.5% FBS in PBS or treated with VEGF-A (50 ng/mL) in 0.5% FBS in PBS for 10 minutes at 37°C. Cells were lysed in 1% Triton X-100 lysis buffer, and equal amounts of protein were Western blotted with anti-p-Y323 Syk, total Syk (supplemental Figure 5A), anti-p-Y1175 VEGFR-2, and total VEGFR-2 Abs, as indicated. The results showed an up-regulation of p-Y323 Syk in response to VEGF-A treatment. (B) Parallel studies were performed using HUVECs that were infected with full-length CD36 or empty (PINCO) vector (supplemental Figure 4B). After treatment of cells with VEGF-A, cells were lysed in 1% Triton X-100, and immunoblotting was performed as indicated. A significant increase in the level of p-Y1175 VEGFR-2 and p-Y323 Syk was only detected in HUVECs with overexpression of CD36. Note that Syk expression is significantly higher in the HUVECs that were engineered to express CD36 (bottom panel, total Syk, and supplemental Figure 4B). These experiments were repeated 3 times independently. (C) Wild-type and TSP-1–null mice were injected with 2 μg of VEGF-A in 100 μL of sterile PBS via the tail vein. After 5 minutes, mice were euthanized and lungs were harvested as described in the immunoblotting section in “Methods.” An equal amount of protein was Western blotted with anti-p-Y323 and anti-total Syk. VEGF-A treatment up-regulated the level of Syk phosphorylation as was seen in panel A, but the response was higher in wild-type mice compared with TSP-1–null, even though the wild-type mice express lower levels of total Syk (bottom panel and supplemental Figure 4A). The slight difference in Syk expression (Figure 4A-B) is possibly because of difference in loading, as it is also detected in actin and VEGFR-2 lanes. Because cells were treated only for 10 minutes, it is not sufficient time for VEGF-A to up-regulate Syk expression. In these experiments, we resuspended cells in PBS with BSA to avoid any interference from the media or serum.

VEGF-A–induced phosphorylation of Syk (Y323). (A) HDMECs were serum-starved overnight in media containing 2% FBS. Cells were washed and incubated with either 0.5% FBS in PBS or treated with VEGF-A (50 ng/mL) in 0.5% FBS in PBS for 10 minutes at 37°C. Cells were lysed in 1% Triton X-100 lysis buffer, and equal amounts of protein were Western blotted with anti-p-Y323 Syk, total Syk (supplemental Figure 5A), anti-p-Y1175 VEGFR-2, and total VEGFR-2 Abs, as indicated. The results showed an up-regulation of p-Y323 Syk in response to VEGF-A treatment. (B) Parallel studies were performed using HUVECs that were infected with full-length CD36 or empty (PINCO) vector (supplemental Figure 4B). After treatment of cells with VEGF-A, cells were lysed in 1% Triton X-100, and immunoblotting was performed as indicated. A significant increase in the level of p-Y1175 VEGFR-2 and p-Y323 Syk was only detected in HUVECs with overexpression of CD36. Note that Syk expression is significantly higher in the HUVECs that were engineered to express CD36 (bottom panel, total Syk, and supplemental Figure 4B). These experiments were repeated 3 times independently. (C) Wild-type and TSP-1–null mice were injected with 2 μg of VEGF-A in 100 μL of sterile PBS via the tail vein. After 5 minutes, mice were euthanized and lungs were harvested as described in the immunoblotting section in “Methods.” An equal amount of protein was Western blotted with anti-p-Y323 and anti-total Syk. VEGF-A treatment up-regulated the level of Syk phosphorylation as was seen in panel A, but the response was higher in wild-type mice compared with TSP-1–null, even though the wild-type mice express lower levels of total Syk (bottom panel and supplemental Figure 4A). The slight difference in Syk expression (Figure 4A-B) is possibly because of difference in loading, as it is also detected in actin and VEGFR-2 lanes. Because cells were treated only for 10 minutes, it is not sufficient time for VEGF-A to up-regulate Syk expression. In these experiments, we resuspended cells in PBS with BSA to avoid any interference from the media or serum.

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