Targeted disruption of the mouse VE-PTP gene. (A) Targeting strategy. An IRES-lacZ-pgk-neo cassette was inserted between VE-PTP exons 17 and 19, resulting in a truncated protein lacking the transmembrane, the cytoplasmic, and the 17th extracellular domains. Exons are given as open rectangles; arrows depict the location of primers for genotyping; CD indicates cytoplasmic domain; and TM indicates transmembrane domain. Drawing is not to scale. (B) Western blot analysis of cell lysates and culture supernatants (as indicated) from embryonic endothelioma cells established from wild-type (VE-PTP^+/+) and homozygous (VE-PTP^mt/mt) VE-PTP mutant embryos. Blots were analyzed with a polyclonal serum against the C-terminus of VE-PTP (VE-PTP-C). (C) Autoradiograph of VE-PTP immunoprecipitations of embryonic endothelioma cells established from wt, heterozygous, or homozygous VE-PTP mutant embryos (as indicated), metabolically labeled with [³⁵S]-methionine/cysteine. Wild-type and truncated mutant forms of VE-PTP were immunoprecipitated with mAb 109.3 directed against the extracellular domain of VE-PTP. (D) FACS analysis showing the surface expression of major endothelial marker proteins of endothelioma cells of wild-type, heterozygous, and homozygous VE-PTP mutant phenotype. Note that homozygous mutant endothelioma cells showed no FACS signal for VE-PTP. Mutation of VE-PTP did not affect the expression of endoglin, Flk-1, ICAM-2, N-cadherin, PECAM-1, Tie-2, or VE-cadherin.