|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 3. EphA2 receptor is required for ephrin-A1-induced endothelial cell migration. (A) Immunoblot analysis of EphA2 expression in wild-type (+/+), heterozygous (+/–) and Ad-EphA2 transduced EphA2-/– MPMEC lysates versus Ad-ßgal transduced EphA2-/– MPMEC lysates. (B) Migration of MPMEC derived from wild-type, heterozygous, or EphA2-deficient mice in response to ephrin-A1 was quantified by transwell assay. EphA2-deficient MPMECs were infected with recombinant adenoviruses encoding ß-galactosidase (EphA2–/–Ad-ßgal) or wild-type EphA2 (EphA2–/–Ad-EphA2) 48 hours prior to migration assay. The number of endothelial cells that had migrated to the lower surface of the transwell were counted. Three fields per transwell were scored for each condition in triplicate samples and data are means±s.d. of three independent experiments. Significant differences in migration for EphA2–/–Ad-EphA2 (*) or Ad-ßgal (**) compared to other experimental conditions are indicated where P<0.01 using ANOVA analysis. (C) Immunoblot analysis of EphA2 immunoprecipitated from BPMEC lysates showing elevated tyrosine phosphorylation of EphA2-NeuTM mutant in the absence of ephrin-A1 stimulation. (D) Immunoblot analysis of immunoprecipitated EphA2 showing decreased tyrosine phosphorylation of endogenous EphA2 after ephrin-A1 stimulation in EphA2-
C-expressing BPMEC relative to mock transfected cells. (E) Migration of BPMEC expressing kinase elevated EphA2 (EphA2-NeuTM) or truncated, dominant negative EphA2 (EphA2-C) in response to ephrin-A1 was also quantified by transwell assay. Significant differences in migration are indicated for P<0.01 using Student's t-test: ***P=0.0001 EphA2-NeuTM +/–ephrin-A1 versus mock unstimulated, ****P=0.0005 EphA2-C + ephrin-A1 versus mock + ephrin-A1. (F) Expression of EphA2-NeuTM or EphA2-C in BPMEC was confirmed by immunoblot analysis.
|
