Bis tris gel

Total cell lysates were prepared from BMDC cultures using Pierce IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol with complete protease inhibitors) and concentration was determined (Pierce B.C.A. kit, ThermoFisher Scientific). Forty micrograms of protein that had been separated by SDS-PAGE using 4-12% Bis-Tris gels (Lonza, Walkersville, MD) was transferred to a nitrocellulose membrane. Membranes were reacted with anti-rabbit Gclc (1:500 dilution) (ThermoFisher Scientific), Irg1, and Sod1 monoclonal antibodies (1: 1000 dilution) (Cell signaling Technology, Danvers, MA) and probed with anti-rabbit β-actin before developing in Odyssey system (Li-COR).

Samples were prepared for Western blot by scraping cells in lysis buffer at 4°C for 20 min. Samples were then centrifuged for 10 min at 10,000g, 4 °C to remove insoluble material. Cell lysates were normalized for protein content, separated by SDS-PAGE on a 4–20% Bis-Tris gel (Lonza), and transferred to a nitrocellulose membrane (Invitrogen, Iblot). After blocking in 5% Blotto (Bioexpress), membranes were incubated with primary peNOS (1179) antibody (Invitrogen) overnight at 4 °C followed by a secondary horseradish peroxidase-conjugated antibody for 2 h at room temperature. Following protein band detection, the membrane was stripped (Western blot stripping buffer, Pierce) for 8 min and then incubated overnight with primary eNOS antibody (BD biosciences) at 4 °C and subsequently by a secondary horseradish peroxidase-conjugated antibody at room temperature. Protein bands were detected using an enhanced chemiluminescence kit (Western Lightning, PerkinElmer), and visualized with a Fluorchem digital imager (Alpha Innotech). Band intensity was quantified using AlphaEase FC software.