Figure 2.
Figure 2. VEGFR2 clustering and internalization induced by soluble and matrix-bound VEGF165. (A) HUVECs were treated with vehicle (−), Vs (200 ng/ml), Vb (200 ng/ml), or collagen (col) for 15 min. Cells were fixed and stained using either rabbit Ig control (top) or antibody against extracellular domain of VEGFR2 in the absence of permeabilization. Computer-generated images (black and white on the right) were used for quantitation of positive staining of VEGFR2 (VEGFR2 distribution) and to identify particle size over 10-fold larger than controls (VEGFR2 clustering). (B) Statistical analysis for VEGFR2 distribution and VEGFR2 clustering are shown. For VEGFR2 clustering, baseline (=1) size was assessed when cells were exposed to vehicle only (*, P < 0.05, between Vb to either −, Vs, or Col). (C) Internalization of VEGFR2 was analyzed in confluent HUVECs treated with Vs (200 ng/ml), Vb (200 ng/ml), and vehicle controls for 15 min. To detect the internalized receptor, cells were treated first with a recombinant single-chain antibody against human VEGFR2 (scFvA7), subsequently exposed to Vs or Vb, followed by acid washes before fixation. Internalized VEGFR2 appears in a vesicular pattern. (D) Same experiments as C but analyzed by Western blots using VEGFR2 or 4G10 antibodies. Total VEGFR2 was measured from the same lysate used for immunoprecipitation used for internalized VEGFR2. (E) Statistics analysis of C. The results (referred to as counts per cell) were reported in the bar graph of three independent experiments. Six random fields were analyzed in each experiment. (F) Densitometric analysis of D. The results were from six independent experiments. Internalized VEGFR2 by Vs and Vb were normalized with total VEGFR2 in the lysate, and then compare normalized Vb over Vs in each experiment. (G) Confluent HUVECs were pretreat with Dynosore or DMSO at 37°C for 15 min. Cells were then treated with Vs, Vb, or vehicles (−) for 5 min. Phosphorylation assays were performed and the cell lysates were then immunoprecipitated with 4G10 and then blotted with total VEGFR2 antibody. Total VEGFR2 was determined by using the same lysate used for immunoprecipitation. In A and C, nuclei stained with TOPRO3 appears in blue. *, P < 0.05; **, P < 0.005.

VEGFR2 clustering and internalization induced by soluble and matrix-bound VEGF165. (A) HUVECs were treated with vehicle (−), Vs (200 ng/ml), Vb (200 ng/ml), or collagen (col) for 15 min. Cells were fixed and stained using either rabbit Ig control (top) or antibody against extracellular domain of VEGFR2 in the absence of permeabilization. Computer-generated images (black and white on the right) were used for quantitation of positive staining of VEGFR2 (VEGFR2 distribution) and to identify particle size over 10-fold larger than controls (VEGFR2 clustering). (B) Statistical analysis for VEGFR2 distribution and VEGFR2 clustering are shown. For VEGFR2 clustering, baseline (=1) size was assessed when cells were exposed to vehicle only (*, P < 0.05, between Vb to either −, Vs, or Col). (C) Internalization of VEGFR2 was analyzed in confluent HUVECs treated with Vs (200 ng/ml), Vb (200 ng/ml), and vehicle controls for 15 min. To detect the internalized receptor, cells were treated first with a recombinant single-chain antibody against human VEGFR2 (scFvA7), subsequently exposed to Vs or Vb, followed by acid washes before fixation. Internalized VEGFR2 appears in a vesicular pattern. (D) Same experiments as C but analyzed by Western blots using VEGFR2 or 4G10 antibodies. Total VEGFR2 was measured from the same lysate used for immunoprecipitation used for internalized VEGFR2. (E) Statistics analysis of C. The results (referred to as counts per cell) were reported in the bar graph of three independent experiments. Six random fields were analyzed in each experiment. (F) Densitometric analysis of D. The results were from six independent experiments. Internalized VEGFR2 by Vs and Vb were normalized with total VEGFR2 in the lysate, and then compare normalized Vb over Vs in each experiment. (G) Confluent HUVECs were pretreat with Dynosore or DMSO at 37°C for 15 min. Cells were then treated with Vs, Vb, or vehicles (−) for 5 min. Phosphorylation assays were performed and the cell lysates were then immunoprecipitated with 4G10 and then blotted with total VEGFR2 antibody. Total VEGFR2 was determined by using the same lysate used for immunoprecipitation. In A and C, nuclei stained with TOPRO3 appears in blue. *, P < 0.05; **, P < 0.005.

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