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Blood, Vol. 107, Issue 12, 4754-4762, June 15, 2006
Vascular endothelial cell–specific phosphotyrosine phosphatase (VE-PTP) activity is required for blood vessel development
Blood Bäumer et al.
107: 4754
Supplemental materials for: Bäumer et al
Files in this Data Supplement:
- Figure S1. Specific expression of VE-PTP on blood vessels in various adult mouse organs (PDF, 249 KB) -
Cryostat sections of mouse intestine, tongue, ovary, heart, and uterus (as indicated) were stained either with a mAb against VE-PTP or a mAb against endomucin (as indicated). VE-PTP was also specifically expressed on endothelium of liver, kidney, lung, testis and brain (not shown). Note that endocard in adult hearts was only weakly stained for VE-PTP.
- Figure S2. Expression of a variety of angiogenesis related receptors and signaling factors is unaffected in VE-PTP gene disrupted mice (PDF, 265 KB) -
RNA isolated from E9.5 wt or VE-PTPmt/mt mouse embryos was analysed by semiquantitative RT-PCR as described below. PCR reactions were carried out under nonsaturating conditions, using 2-fold serial dilutions of the input cDNA or for negative controls non reverse-transcribed RNA (-RT). PCR products were electrophoresed and detected with ethidium bromide.
Method: Total RNA from whole embryos was isolated using an RNA isolation kit (Qiagen) and DNA was removed using the DNase I Kit (Qiagen). First-strand cDNAs were generated in the presence of RNAsin (Promega), using Super Script II Reverse Transcriptase (Invitrogen) and random hexamer primers (Amersham) according to manufacturer’s conditions. Control reactions without reverse transcriptase were performed for each RNA sample. PCR reactions were done under linear conditions, allowing to detect increasing amounts of product with increasing PCR cycles. PCR primers for the various genes are given in the table below. First-strand cDNAs (31 ng) were amplified in a final volume of 20 µl with 0.3 U Go-Taq DNA polymerase (Invitrogen) and 13 pmol of each nucleotide primer. For Tie-2 and Ang-1, the amplification parameters were 10 min at 95°C, followed by 34 to 35 cycles of 95°C for 15 sec. and 60°C for 1 min. For all other genes, the amplification parameters were 94°C for 4 min followed by 31 to 36 cycles of 94°C for 30 sec., 60°C for 45 sec., and 72°C for 1 min. n.d. not determined.
Cited References for Figure S2:
Morisada T., Oike Y., Yamada Y, Urano T., Akao M., Kubota Y., Maekawa H., Kimura Y., Ohmura M., Miyamoto T., Nozawa S., Young Koh G., Alitalo K., and Suda T. (2005). Angiopoietin-1 promotes LYVE-1–positive lymphatic vessel formation. Blood 105, 4649-56.
Thijssen VL, Brandwijk RJ, Dings RP, Griffioen AW. (2004). Angiogenesis gene expression profiling in xenograft models to study cellular interactions. Exp Cell Res. 299(2), 286-93.
Vittet D., Prandini M-H., Berthier R., Schweizer A., Martin-Sisteron H., Uzan G. and Dejana E. (1996). Embryonic stem cells differenciate in vitro to endothelial cells through successive maturation steps. Blood 88, 3424-3431.
- Figure S3. Ultrastructural analysis of E9.0 yolk sac endothelium (PDF, 999 KB) -
Ultrathin sections of E9.0 wt (A) and VE-PTPmt/mt (B) yolk sacs were analysed by electron microscopy. The endothelium (EN) lines a blood vessel in the wt yolk sac that is embedded between mesothelium (ME) and extraembryonic endoderm (EN). Due to the large blood lacuna formed in VE-PTPmt/mt yolk sacs the electron micrograph only shows endoderm-associated endothelium. Note the intact tight junctions (marked by arrows) between endothelial cells in the wt as well as in the mutant yolk sacs.
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