Fuji las 4000 luminescent image analyzer

Protein expression was determined by Western immunoblot analysis per the technique outlined previously [4 (link)]. The 20 µg samples of soleus were loaded on 10% polyacrylamide gels and electrophoresed using a Bio-Rad Protein III gel-box onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Soleus extracts (20 µg) along with sample buffer were loaded into wells of 10% SDS-PAGE gels. Samples were then electrophoresed at 120 V for 75 min. Membranes were blocked in a non-fat milk buffer (5% non-fat milk in TBS) for 1 h. Following blocking, membranes were incubated overnight (4 °C) in blocking buffer with the appropriate primary antibodies: anti-MnSOD (rabbit pAb; 1:20,000; Cat# ab13533, Abcam, Cambridge, MA, USA), anti-Nrf2 (rabbit pAb; 1:1000; Cat# 16396-1-AP, Proteintech, Rosemont, IL, USA), anti-HSP70 (rabbit pAb; 1:2000; Cat# ADI-SPA-812-F, Enzo, Farmingdale, NY, USA), and anti-HSP90 (rabbit mAb; 1:1000; Cat# C45G5, Cell Signaling, Danvers, MA, USA).
Proteins were visualized by Super Signal West Dura Extended Duration Substrate (Cat# 34076, Thermo Scientific, Waltham, MA, USA) enhanced chemiluminescence detection, and developed with a Fuji LAS-4000 Luminescent Image Analyzer (FujiFilm Medical Systems, Lexington, MA, USA). Quantification was performed using NIH ImageJ software with Ponceau-S staining (cytoplasmic: band ~38 kDa mark (GAPDH)) used as a loading control.

Using Jurkat cell membranes prepared as described [41 (link)], a polyclonal antibody directed at Gα_s residues 28–42 [42 (link)], prepared in the laboratory of Henry Bourne (University of California, San Francisco), was used to detect expression of Gα_s, and Gβ₁ (XAB-00301-1-G) and Gβ₂ (XAB-00401-1-G) antibodies from CytoSignal, LLC were used to detect expression of Gβ₁ and Gβ₂, respectively. Membrane proteins were resolved on NuPAGE 4–12% Bis-Tris gels and transferred to Invitrolon PVDF membranes (Life Technologies). The antigen-antibody complexes were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc.). Chemiluminescence was imaged using a Fuji LAS-4000 Luminescent Image Analyzer. Bands in the images were quantified using ImageJ software. For quantification of Gα_s, both the long and short forms of Gα_s [43 (link)] were measured together.

THP-1 monocytes were washed in cold PBS and lysed for 15 min in ice-cold lysis buffer (20 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM MgCl₂, 1% n-dodecyl-(β)-d-maltoside, 50 mM sodium fluoride, and a protease inhibitor cocktail, all purchased from Sigma-Aldrich). Protein concentration was quantified with the NanoDrop-1000 Spectrophotometer (Thermo Fisher Scientific). Cell lysates were resolved on NuPAGE Bis-Tris gels and transferred to nitrocellulose membranes. Protein detections used the anti-FADS2 and anti-GAPDH antibodies. Protein complexes were revealed with the ECL Prime detection reagent (GE Healthcare) and chemiluminescence reading on a Fuji LAS-4000 Luminescent Image Analyzer.