Figure 6
Figure 6. DEP-1 promotes VEGF-induced Cortactin tyrosine phosphorylation and endothelial cell invasion through Y1311 and Y1320. (A) HUVECs were transfected with control (CTL) and DEP-1 siRNAs and then stimulated with VEGF (50 ng/mL) as indicated in legend to Figure 5A. Cortactin tyrosine phosphorylation was monitored by immunoblotting total cell lysates (TCL) with the pY421Cortactin antibody. DEP-1 expression and Cortactin protein levels were detected with the DEP-1 goat and Cortactin (4F11clone) antibodies, respectively. (B) BAECs were transfected with WT and mutant DEP-1 constructs, serum-starved, and then stimulated 5 minutes with VEGF (50 ng/mL). Cortactin tyrosine phosphorylation and protein levels were monitored as described in panel A. (C) DEP-1 is required for the localization of Cortactin in membrane protrusions. HUVECs transfected with control (CTL) or DEP-1siRNAs were plated on gelatin-coated glass coverslips, serum-starved, and then stimulated with VEGF (10 ng/mL) for 5 minutes before cell fixation. Immunostaining with Cortactin (4F11 clone) and Alexa Fluor 594–coupled mouse secondary antibodies is shown. The percentage of cells with enriched localization of Cortactin in membrane protrusions was evaluated in control and DEP-1–depleted cells in 4 microscopic fields at ×40 magnification; *P < .05. (D) HUVECs were transfected with control (CTL) or DEP-1 siRNAs and seeded in duplicates 48 hours after transfection on Transwell filter inserts previously coated with 50 μL of Matrigel (2 mg/mL). Cells were allowed to invade for 24 hours, and then processed to visualize and count cells on the lower side of the filter. Results ± SD have been normalized using the average from the control condition; *P < .05. (E) HUVECs transfected with empty vector (pmT2), WT DEP-1, DEP-1 Y1311F/Y1320F (YY/FF), or DEP-1 C/S were plated in duplicates on Matrigel-coated filter inserts and processed as described in panel D. Results are representative of 5 independent experiments; *P < .05. All other results shown in this figure are representative of 3 independent experiments.

DEP-1 promotes VEGF-induced Cortactin tyrosine phosphorylation and endothelial cell invasion through Y1311 and Y1320. (A) HUVECs were transfected with control (CTL) and DEP-1 siRNAs and then stimulated with VEGF (50 ng/mL) as indicated in legend to Figure 5A. Cortactin tyrosine phosphorylation was monitored by immunoblotting total cell lysates (TCL) with the pY421Cortactin antibody. DEP-1 expression and Cortactin protein levels were detected with the DEP-1 goat and Cortactin (4F11clone) antibodies, respectively. (B) BAECs were transfected with WT and mutant DEP-1 constructs, serum-starved, and then stimulated 5 minutes with VEGF (50 ng/mL). Cortactin tyrosine phosphorylation and protein levels were monitored as described in panel A. (C) DEP-1 is required for the localization of Cortactin in membrane protrusions. HUVECs transfected with control (CTL) or DEP-1siRNAs were plated on gelatin-coated glass coverslips, serum-starved, and then stimulated with VEGF (10 ng/mL) for 5 minutes before cell fixation. Immunostaining with Cortactin (4F11 clone) and Alexa Fluor 594–coupled mouse secondary antibodies is shown. The percentage of cells with enriched localization of Cortactin in membrane protrusions was evaluated in control and DEP-1–depleted cells in 4 microscopic fields at ×40 magnification; *P < .05. (D) HUVECs were transfected with control (CTL) or DEP-1 siRNAs and seeded in duplicates 48 hours after transfection on Transwell filter inserts previously coated with 50 μL of Matrigel (2 mg/mL). Cells were allowed to invade for 24 hours, and then processed to visualize and count cells on the lower side of the filter. Results ± SD have been normalized using the average from the control condition; *P < .05. (E) HUVECs transfected with empty vector (pmT2), WT DEP-1, DEP-1 Y1311F/Y1320F (YY/FF), or DEP-1 C/S were plated in duplicates on Matrigel-coated filter inserts and processed as described in panel D. Results are representative of 5 independent experiments; *P < .05. All other results shown in this figure are representative of 3 independent experiments.

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