Anti phospho ampka

The mashed tissues and cells were lysed in RIPA lysis buffer (150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Igepal, 50 mM Tris-HCl pH 8.0 and 2 mM EDTA) in an ice bath for 30 min and centrifuged at 18,000 × g rpm for 30 min at 4°C. The supernatant was stored at −80°C. A BCA kit was used to determine the protein concentration. The same amount of protein (8 µg) was separated via 10% SDS-PAGE and transferred to a PVDF membrane. After the PVDF membrane was blocked in 5% BSA (cat. no. 9048-46-8; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 1 h, it was incubated with anti-SIRT1 (cat. no. 19A7AB4; 1:2,000; Abcam), anti-AMPK (cat. no. D63G4; 1:1,000; Cell Signaling Technology, Inc.), anti-phospho-AMPKa (cat. no. D4D6D; 1:1,000; Cell Signaling Technology, Inc.) and anti-β-actin antibodies (cat. no. T0022; 1:3,000; Affinity Biosciences) overnight at 4°C. Subsequently, it was incubated with HRP-conjugated goat anti-rabbit IgG (cat. no. S0001) and goat anti-mouse IgG (cat. no. S0002) secondary antibodies (both 1:10,000; Affinity Biosciences) at room temperature for 1 h. The color of the PVDF membrane was developed using an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.), and the gray value was analyzed using ImageJ software (version 1.8.0; National Institutes of Health).

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.