Journal:

Article Title: Dissecting the role of endothelial SURVIVIN ?Ex3 in angiogenesis

doi: 10.1182/blood-2006-02-003749

Figure Lengend Snippet: Survivin ΔEx3 is involved in Rac1 activation during endothelial tube formation. (A) HUVECs plated onto BME were analyzed for Rac1 activation at the tube formation stage (4 hours) by GST-PBD pull-down assays. The amount of active Rac1 was normalized against total Rac1 in the corresponding lysate, and the integrated density values of the bands were measured using Syngene GeneTools analysis software. (B-H) HUVE cells were plated on growth factor–reduced BME in 96-well plates supplemented with VEGF (20 ng/mL) or Rac1 inhibitor (100 μM), untransfected, or after transfection with eGFP Survivin ΔEx3. (B) Control; (C) control + VEGF; (D) GFP Survivin ΔEx3; (E) Survivin ΔEx3 antibody (10 μg/mL); (F) control + Rac1 inhibitor; and (G) GFP Survivin ΔEx3 + Rac1 inhibitor. Tube formation was assayed at 4 hours after plating on BME, and quantitative data were collected by measuring the length of the tubules formed under an inverted light microscope at ×100 magnification (40×/0.75 objective lens). Quantitative data shown in panel H are mean ± SE of 5 replicates per sample.

Article Snippet: The amount of active Rac1 was normalized against total Rac1 in the corresponding lysate, and the integrated density values of the bands were measured using Syngene GeneTools analysis software. (B-H) HUVE cells were plated on growth factor–reduced BME in 96-well plates supplemented with VEGF (20 ng/mL) or Rac1 inhibitor (100 μM), untransfected, or after transfection with eGFP Survivin ΔEx3. (B) Control; (C) control + VEGF; (D) GFP Survivin ΔEx3; (E) Survivin ΔEx3 antibody (10 μg/mL); (F) control + Rac1 inhibitor; and (G) GFP Survivin ΔEx3 + Rac1 inhibitor.

Techniques: Activation Assay, Software, Transfection, Light Microscopy