Ag 27b 0016pf

For ANG-2 antibody neutralization experiments, antibody was supplied by AdipoGen, which is a mouse IgG2bλ (anti-Angiopoietin-2, mAb (rec.) (blocking) (Angy-2–1) (AG-27B-0016PF, AdipoGen Life Sciences, RRID: AB_2490509). Anti ANG-2 antibody (ab8452, RRID: AB_306569) for Western blotting and immunofluorescence staining was purchased from Abcam (Cambridge, MA). An antibody against β-actin (A3854, RRID: AB_262011) was from Sigma-Aldrich (St. Louis, MO). Anti-endomucin (sc-65495, RRID: AB_2100037) and anti F4/80 (sc-25830, RRID: AB_2246477) primary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Perilipin antibody (20R-PP004) was obtained from Fitzgerald, Inc. (Acton, MA). Anti-HIF-1α primary antibody (NB100-105, RRID: AB_10001154) was purchased from Novus Biologicals (Littleton, CO). Primary antibodies against GAPDH (#2118S, RRID: AB_561053), phosphorylated Akt (pAkt, Ser473, #9271S, RRID: AB_329825) and total Akt (#2920S, RRID: AB_1147620) came from Cell Signaling Technology, Inc. (Beverly, MA). Unless specifically indicated, all other reagents were obtained from Sigma-Aldrich Corporation (St. Louis, MO).

For prophylactic AA experiments, 2 mg of AA (Fisher Scientific: #ICN19462510) was diluted in 30 μL of corn oil (Sigma C8267) which was administered 4–6 hours prior to CS challenge via oral gavage. Control animals received 30 μL corn oil alone. All other experiments used saline controls. For exogenous Ang1 and Ang2 experiments, 500 ng of Ang1 or Ang2 (R&D Systems: #9936-AN and #7186-AN respectively) was administered via intraperitoneal injection 1 hour prior to CS challenge. For anti-Angiopoietin-2 antibody treatment 50 μg of antibody (Adipogen: #AG-27B-0016PF) was administered concurrently with CS challenge via intraperitoneal injection. For L-NAME experiments, 10 μg of L-NAME (Abcam: #120136) reconstituted in distilled water was injected via intraperitoneal injection concurrently with CS challenge. Finally, for L-Arginine experiments, 5 mg of L-Arginine (Cayman Chemical: #23703) reconstituted in distilled water was administered via intraperitoneal injection 4-6 hours prior to CS challenge. The schedules described above were maintained for all combinational interventions described.