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Figure 3 | BMC Biochemistry

Figure 3

From: Src Kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells

Figure 3

GST pull-downs and Far Western blots indicate increased Src SH3-SH2 interaction with KDR/Flk-1 upon VEGF stimulation. (A) HUVECs were either serum-starved (0') or starved and then VEGF-treated (15 ng/mL for 15 min) (15'). The indicated GST-Src domain fusion proteins (see below) bound to glutathione-agarose beads were used (500 pmol equimolar amounts each), in pull-down incubations with equal amounts of protein lysate samples (500 μg each). The pull-down complexes were resolved on a 7% acrylamide SDS-PAGE gel and transferred to nitrocellulose. Immunoblotting was then performed using α-KDR/Flk-1 (A-3) or α-Flt-1 (H-225). GU, GST-Src unique domain fusion protein; GU32, GST-Src unique SH3 SH2 domain fusion protein; G32, GST-Src SH3 SH2 domain fusion protein. (B) Cell extract (pre-pull-down) input levels of phosphotyrosine KDR/Flk-1 and KDR/Flk-1, from HUVECs either serum-starved (0') or starved and then VEGF-treated (15'). (C) Cell extract (pre-pull-down) input levels of phosphotyrosine Flt-1 and Flt-1, from HUVECs either serum-starved (0') or starved and then VEGF-treated (15'). (D) HUVECs were serum-starved (-) or VEGF-treated with VEGF (15 ng/mL for 15 min) (+). The cell extracts were then immunoprecipitated using α-KDR/Flk-1 (N-931). The immunocomplexes were resolved on a 7% acrylamide SDS-PAGE gel. Biotinylated GST and biotinylated GST-Src SH3-SH2 fusion protein were used in Far Western blotting. Detection was performed using horseradish peroxidase-conjugated ExtrAvidin®, followed by enhanced chemiluminescence detection reagents. Immunoprecipitation-Western blots for levels of phosphotyrosine KDR/Flk-1 and KDR/Flk-1 of the serum-starved (-) or VEGF-treated HUVEC (+) cell extracts were also performed in parallel.

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