Figure 4.
Figure 4. VEGF-induced activation of Src and the effect of tsad silencing. (A) NanoPro analysis of c-Src posttranslational modification in HDMECs was performed on cells treated with vehicle (basal) cells or with VEGF for 7 min. Cell lysates were subject to capillary isoelectric focusing and probing with anti–c-Src pY418, anti–c-Src pY527, and anti–c-Src protein antibodies. Peaks (P) identified by antibody-binding, followed by enhanced chemiluminescence are indicated. Bottom panels show antibody detection of c-Src protein after phosphatase treatment. Representative results of three independent experiments are shown. (B) Quantification of one experiment as shown in A. (C) HDMECs were transfected with control siRNA (si ctr) or different tsad siRNAS followed by stimulation with VEGF for 7 or 15 min. Blotting was performed on total lysates with antibodies against TSAd; phosphorylated proteins c-Src pY418, pY1175 (autophosphorylation site in VEGFR2), pPLCγ, peNOS, pERK1/2; and against the corresponding total protein pools. Molecular weights in kilodaltons (kD) are indicated to the right. Immunoblotting for β2 microglobulin was used as a control for equal loading. All results are from the same set of extracts run out on different gels to optimize separation. Numbers under total c-Src bands indicate fold change in c-Src pY418/total c-Src protein in the different conditions; the c-Src pY418/total c-Src ratio in the unstimulated ctr-siRNA/ sample was set to 1. Representative results of at least three independent experiments are shown. (D) Immunofluorescent staining for VE-cadherin (white), c-Src pY418 (red), and paxillin (green) of HDMEC cultures transfected with control siRNA (si ctr) or tsad siRNA1 (si tsad) followed by stimulation with VEGF for 7 min. Bottom panels show magnification of merged images from boxed regions in the middle row of panels. White line outlines VE-cadherin–positive junctions, arrowheads indicate c-Src p418 localized at adherens junctions in control siRNA cells and in focal adhesions in tsad siRNA cells. Representative results of at least three independent experiments are shown. Bar, 20 µm.

VEGF-induced activation of Src and the effect of tsad silencing. (A) NanoPro analysis of c-Src posttranslational modification in HDMECs was performed on cells treated with vehicle (basal) cells or with VEGF for 7 min. Cell lysates were subject to capillary isoelectric focusing and probing with anti–c-Src pY418, anti–c-Src pY527, and anti–c-Src protein antibodies. Peaks (P) identified by antibody-binding, followed by enhanced chemiluminescence are indicated. Bottom panels show antibody detection of c-Src protein after phosphatase treatment. Representative results of three independent experiments are shown. (B) Quantification of one experiment as shown in A. (C) HDMECs were transfected with control siRNA (si ctr) or different tsad siRNAS followed by stimulation with VEGF for 7 or 15 min. Blotting was performed on total lysates with antibodies against TSAd; phosphorylated proteins c-Src pY418, pY1175 (autophosphorylation site in VEGFR2), pPLCγ, peNOS, pERK1/2; and against the corresponding total protein pools. Molecular weights in kilodaltons (kD) are indicated to the right. Immunoblotting for β2 microglobulin was used as a control for equal loading. All results are from the same set of extracts run out on different gels to optimize separation. Numbers under total c-Src bands indicate fold change in c-Src pY418/total c-Src protein in the different conditions; the c-Src pY418/total c-Src ratio in the unstimulated ctr-siRNA/ sample was set to 1. Representative results of at least three independent experiments are shown. (D) Immunofluorescent staining for VE-cadherin (white), c-Src pY418 (red), and paxillin (green) of HDMEC cultures transfected with control siRNA (si ctr) or tsad siRNA1 (si tsad) followed by stimulation with VEGF for 7 min. Bottom panels show magnification of merged images from boxed regions in the middle row of panels. White line outlines VE-cadherin–positive junctions, arrowheads indicate c-Src p418 localized at adherens junctions in control siRNA cells and in focal adhesions in tsad siRNA cells.. Representative results of at least three independent experiments are shown. Bar, 20 µm.

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