Immunoblot analysis with membrane fractions show that thrombin treatment decreased Occludin distribution in membrane fraction in time a dependent manner. (BBB) breakdown and increased endothelial permeability is usually a hallmark of neuro-vascular inflammation. Angiopoietin-1 (Ang-1), a Tie-2 receptor agonist ligand, is known to modulate barrier function of endothelial cells; however the molecular mechanisms related to Ang-1 mediated repair of Tight Junctions (TJs) in brain endothelium still remain elusive. In this study, we investigated a novel role of non-receptor protein tyrosine phosphatase N-2 (PTPN-2) in Ang-1 mediated stabilization of tight junction proteins. Method and Result To study the barrier protective mechanism of Ang-1, we challenged human brain microvascular endothelial cells ECs permeability assay and study Ang-1 functions to counteract deleterious effects of thrombin. We also propose to investigate whether Ang-1 requires PTPN-2 to promote the conversation of Occludin with ZO-1 and its junctional localization to stabilize brain endothelium after thrombin challenge. Materials and Methods Antibodies and chemicals Human r-Ang-1 was purchased from R&D Systems (Minneapolis, MN). Thrombin was purchased from Enzyme Research Laboratories (South Bend, IN). Rabbit monoclonal anti-PTPN-2 antibody was from OriGene Technologies, Inc (Rockville, MD, USA). Rabbit polyclonal anti-Claudin-5, mouse monoclonal anti-PY-20 (clone 4G10) antibody was purchased from Millipore (Billerica, MA). Mouse monoclonal anti-ZO-1, rabbit polyclonal anti-Occludin antibody and 25,26-Dihydroxyvitamin D3 DAPI were purchased from Invitrogen (Life Technologies, Grand Island, NY, USA). Rabbit polyclonal anti-Tie-2 antibody and protein A/G beads were purchased from Santa Cruz Biotechnology Inc (Dallas, TX, USA). Mouse anti-Na+-K+ ATPase 3 was from B.D. Biosciences (San Jose, CA). Rabbit anti phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), anti- p44/42 MAPK (Erk1/2) and anti actin was from Cell Signaling Technology (Danvers, MA). FITC conjugated secondary antibody and affinipure goat anti rabbit polyclonal light chain specific secondary antibody was from Jackson ImmunoResearch (West Grove, PA). ON-TARGET plus human PTPN-2 siRNA (ID: J-008969-05-0005) and mismatch control, transfection reagents were purchased from Dharmacon. All other chemicals were obtained from Sigma-Aldrich (St Louis, MI, USA). Cell culture, treatment and transfection Human brain micro-vascular endothelial cells (HBMECs) were cultured and produced as published earlier by Stins et al. 1997 . For Ang-1 and thrombin treatments, ECs were starved in RPMI-2 supplemented with 0.2% FBS for 4 hr. 70C80% confluent monolayer of ECs were transfected using siRNA transfection reagent from Dharmacon in accordance with the manufacturers instructions. Isolation of sub cellular fractions After thrombin or Ang-1/thrombin treatment, ECs monolayer was washed with PBS, scraped and pelleted by centrifugation at 1000for 5 min. Sub cellular fractions were isolated by using Subcellular Protein Fractionation Kit for cultured cells from 25,26-Dihydroxyvitamin D3 Thermo Scientific (Waltham, MA). Co-immunoprecipitation and western blot analysis ECs were lysed in altered RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Sodium Deoxycholate, 1% Triton X-100, 25,26-Dihydroxyvitamin D3 1 mM Na3VO4, 0.1% SDS, 10 g/ml Aprotinin, 10 g/ml Leupeptin and 100M PMSF). For co-immunoprecipitation experiments, after treating ECs with agonists, 350 g of total cellular protein was incubated with 2.5 g of primary antibody overnight at 4C, and precipitates were collected with 30l of protein A/G agarose beads. Western blotting was performed as described earlier . Molecular mass standards are indicated next to each blot in kilodaltons. Each immunoprecipitation assay was standardized with respect to their IgG control as shown in S1 Fig. permeability assay The permeability of HBMECs monolayer to Evans Blue labeled Albumin (EBA) was decided as described . ECs were produced to 25,26-Dihydroxyvitamin D3 confluence on transwell inserts with 0.4-m pore size (Corning transwell polyester membrane cell culture inserts cat. Rabbit Polyclonal to BAIAP2L2 no. CLS3460). Upper (luminal) and lower (abluminal) chambers were filled with HBSS made up of 0.5% BSA and 20mM 25,26-Dihydroxyvitamin D3 HEPES. After 30 min equilibration period, the luminal chamber was loaded with 0.057% EBA. Samples from the abluminal chamber were collected every 15 min for 120 min after 100nM of thrombin treatment. To determine the barrier protective effects of Ang-1, the endothelial monolayer.