Anti nox2 gp91phox

Western Blot was used to detect specific proteins in lysates of MRA tissue as previously described.34 (link) Mice were sacrificed then MRA were immediately harvested and frozen in liquid nitrogen and stored at −80°C. Tissue lysates were prepared by homogenizing in ice cooled Tissue protein Extraction Reagent (Prod# 78510, Thermo Scientific, MA, USA), sonicated for 5 seconds and centrifuged for 15 min at 13,000 rpm. Protein quantification was performed according to Pierce™ BCA Protein Assay Kit (Product No. 23225, Thermo scientific, MA, USA). Specific antibodies against Anti-Phospho-Akt (ser473, Cat #9271, Cell Signaling, MA, USA), Anti-total-Akt (Cell Signaling, #9272), Anti-Bip (C50B12, Cell Signaling, #3177), Anti-CHOP (Cell Signaling, #2895), Anti-Phospho-AMPKa (Thr172, Cell Signaling, #2351), Anti-AMPKα (Cell Signaling, #2352), Anti-Phospho-eNOS (Ser1177, Cell Signaling, #9571), Anti-NOX2/gp91phox (Cat #ab80508, Abcam, MA, USA), Anti-NADPH oxidase 4 (#ab133303), Anti-eNOS/NOS Type III (Cat #610296, BD biosciences, CA, USA) and β-actin (Santa Cruz Biotechnology, TX, USA) were purchased and used. All dilutions were prepared according to manufacturer recommendations. Membranes were developed using odyssey-imaging system (LICOR, NE, USA), and band quantification was performed using image J software. Data are expressed after normalization to β-actin as % compared to control.