|
|
Blood, Vol. 109, Issue 4, 1479-1489, February 15, 2007
Dissecting the role of endothelial SURVIVIN  Ex3 in angiogenesis
Blood Caldas et al.
109: 1479
Supplemental materials for: Caldas et al, Vol 109, Issue 4, 1479-1489
Files in this Data Supplement:
- Table S1. Statistical analysis of tube length (PDF, 73.1 KB) -
HUVECs treated with shRNAs and/or antibodies, as detailed in Figure 5. Statistical significance between each treatment condition was evaluated by Student t test and P values represented in the table. * indicates significance (P < .05).
- Figure S1. Survivin ΔEx3 is expressed in brain tumor, but not normal brain (JPG, 32 KB)
-
Protein extracts were prepared as described from normal ependyma, from fresh-frozen biopsies of malignant ependymoma, and from HeLa cells transfected with a myc-tagged survivin  Ex3 construct. One hundred and fifty micrograms (normal ependyma and ependymoma) or 10 µg protein lysate (HeLa cells) were separated by SDS-PAGE and Western blotting was performed as described in “Materials and methods” using a survivin Ex3 antibody. A single band of the expected size was visualized in the ependymoma and HeLa cell (survivin Ex3-myc) lanes.
- Figure S2. Homology of the peptide used to raise the survivin ΔEx3 antibody and a portion of human protein phosphatase 1 (PP1) protein (JPG, 11.4 KB)
-
Homologous amino acids are represented in red.
- Figure S3. Staining patterns of human PP1 and survivin ΔEx3 (JPG, 433 KB)
-
Staining of a representative hemangioma sample for human PP1 and survivin Ex3 shows a different pattern of expression for the 2 proteins. Red staining represents areas of positive reactivity for each of the antibodies. Arrows represent areas of PP1 nuclear staining that are negative for survivin Ex3.
- Figure S4. The survivin ΔEx3 antibody is specific for survivin ΔEx3 protein (JPG, 133 KB)
-
HUVECs were transfected with either scrambled shRNA, survivin Ex3 shRNA, or pan-PP1 siRNA and subjected to immunofluorescence with PP1 antibody (green, Alexa Fluor 488) or survivin Ex3 antibody (red, Texas Red). Cells were counterstained with Hoechst dye (blue) and images acquired by confocal microscopy. In the absence of PP1 protein, the survivin Ex3 antibody still detects survivin Ex3 in a similar pattern as shown in Figure 3. Scale bars represent 20 µm.
- Figure S5. VEGF induces survivin (JPG, 80.5 KB)
-
HUVECs were treated with 20 ng/mL VEGF for 24 hours and protein lysates prepared as described. Fifty micrograms protein were separated by SDS-PAGE and Western blotting was performed as described in “Materials and methods” using a survivin antibody.
- Figure S6. Viability of HUVECs following Rac1 inhibition (JPG, 12.4 KB)
-
HUVECs were incubated with 100 µM in-solution Rac1 inhibitor for 24 hours and cell viability was analyzed by MTT assay. Results reflect the average of 3 experiments, and are represented in comparison with control-treated HUVECs.
|
|
