Figure 9. Hypoxia increases the expression of VEGF receptors and γ-secretase along with VEGFR dissociation from AJ proteins.

(a) Confluent retinal microvascular endothelial cells were exposed to hypoxia for up to 24 hours. Cell lysated were examined by Western blot for VEGFR1, VEGFR2, presenilin-1 (PS-1), nicastrin (NCT), VE-cadherin and β-catenin. α-tubulin acted as the house keeping control. A representative Western blot is shown together with densitometric analysis from three separate experiments. (b) Confluent retinal microvascular endothelial cells were maintained under standard incubator conditions, retinal normoxia and hypoxia for 24 hours. Total cell lysates were split into four equal portions and immunoprecipitated with anti-VE-cadherin, anti-β-catenin, anti-VEGFR1-C-terminus and anti-VEGFR2-C-terminus, and then subsequent Western blot analyzed using these four antibodies, respectively. A panel of representative Western blots is shown. Band intensities of replicate experiments (n = 4) were quantified as described in the Methods and regression analysis undertaken to assess the association of these proteins.