β-catenin dissociates from VEC in response to Hcy. Brain microvascular ECs were cultured and treated beginning 3 days after confluence. Cells were treated with Hcy (20μM) or vehicle control (PBS) for 3 days to mimic a chronic, mild elevation in Hcy. (A) β-catenin associates with the detergent-insoluble, cytoskeletal fraction. To determine the subcellular localization of β-catenin, it was necessary to run pilot experiments to show prevalence of β-catenin in different fractions obtained when applying Nonidet P-40 lysis. Cells were grown to confluence and maintained for 3 days; then a 1% Nonidet P-40 lysis buffer was applied. The portion obtained from this solution was called “extractable” and was centrifuged at 19 000g for 2 hours and then further categorized into “soluble” (supernatant) and “insoluble” (pellet). The fraction of cellular material that remained adherent to the culture plate was solubilized with an ionic detergent lysis buffer (SDS and deoxycholic acid) and called the “non-extractable” fraction. Blots were probed for β-catenin (1:1000, CP1061; ECM Biosciences). (B) Immunohistochemistry demonstrates localization of VEC and β-catenin at cell borders. Hcy had little effect on VEC expression but substantially reduced expression of β-catenin. Scale bar represents 30 μm. (C) Western blot confirms lack of change in VEC but (D) significant loss of β-catenin after Hcy treatment.