8 4 chlorophenylthio cpt camp

The mAbs hec1 (anti-VE-cadherin), hec7 (anti-PECAM-1), and hec2 (anti-CD99) were produced from hybridomas generated in the laboratory as previously described (32 (link)). P1.1 (non-blocking anti-PECAM mAb) was column purified on protein A-Sepharose (GE Healthcare) from ascites provided by P.J. Newman (Blood Center of Wisconsin, Milwaukee, WI) (33 (link)). Fab fragments of P1.1 were cut using papain (Thermo Fisher Scientific), followed by column purification on protein A-Sepharose. Purity of Fab fragments was confirmed by SDS-PAGE. Unlabeled and Dylight 549 conjugated goat-anti-mouse F(ab’)₂ antibodies used for targeted recycling experiments of PECAM-1 were purchased from Jackson ImmunoResearch laboratories. Antibodies for immunofluorescence were conjugated to DyLight 488, DyLight 550 or Alexa Fluor 650 according to the manufacturers protocol (Thermo Fisher Scientific). Alexa Fluor 488 conjugated monoclonal antibodies against occludin and claudin-5 were purchased from Life Technologies (Invitrogen). Polyclonal antibody against claudin-3 was purchased from Life Technologies (Invitrogen). 8-(4-chlorophenylthio(CPT)-cAMP was purchased from Sigma Aldrich and phosphodiesterase inhibitor type IV RO-20–1724 was purchased from Tocris Bioscience.

Astrocytes were grown on Poly-D-lysine coated tissue culture dishes and used until passage 10. ACM was collected and treated as previously described (35 (link)–37 (link)). Briefly, astrocyte cultures were grown at 37°C in a humidified atmosphere of 5% CO₂ and were fed with fresh astrocyte complete medium (ScienCell Research Laboratories) containing low concentration of fetal bovine serum (2%). After ~48h when confluent, the medium was collected, sterile filtered (0.2 µm), centrifuged and diluted 1:1 with HUVEC media and either used immediately or stored at −80°C. HUVEC used for the BBB model were cultured until confluence in ACM. Once confluent, ACM was then supplemented with 17.5 µM RO-20–1724 (Tocris Bioscience) and 250 µM 8-(4-chlorophenylthio(CPT)-cAMP (Sigma Aldrich) for 24h. After 24h the monolayers were extensively washed immediately prior to experimentation. In some experiments, TNF was added to some monolayers to activate endothelial cells. TNF treatment was 20 ng/ml for 4 h, except for Supplemental Fig. 2, where treatments are described in the figure legend.

Ketamine is from KetaSet® and Xylazine from AnaSed®. Medium 199, HBSS, EGTA, HEPES, PenStrep and Gentamycin are from Life Technology. Collagen type 4 is pursached from Worthington. Insulin (Humulin® R U-100) was purchased from Eli Lilly. 8-(4-chlorophenylthio) (CPT)-cAMP, dexamethasone, cycloheximide, Bovine Serum Albumin (BSA), D-glucose and sodium pyruvate were from Sigma-Aldrich. Lipofectamine2000 and Stealth RNAi^™ siRNA against Sin3a were from Thermo Fisher. Trichostatin A (TSA), TMP269 and FK228 were from Selleckchem. TC-H 106 was from Cayman chemical. Insulin was diluted in sterile water. 8-(4-chlorophenylthio) (CPT)-cAMP, D-glucose, sodium pyruvate were dissolved in sterile water. dexamethasone and cycloheximide were dissolved in ethanol (100%). TSA, TMP195, FK228, TC-H 106 were dissolved in DMSO. Anti-FOXO1 (for Western Blot and co-immunoprecipitation, C29H4), anti-SIN3A (for Western Blot, D1B7), and anti-HNF4A (for Western Blot, C11F12) antibodies were from Cell Signaling. Anti-HNF4A ChIP Grade (ab41898), anti-FOXO1A ChIP Grade (ab39670), anti-SIN3A ChIP Grade (ab3479), anti-H3 (ab1791), anti-Ac H3 (ab47915), anti-actin (ab8227) and control IgG antibodies were from Abcam. GCK antibody was from Dr. Magnuson (Vanderbilt University School of Medicine, Nashville, TN).