Runblue sds gel

Cells isolated by centrifugation (600 xg, 2 min) were lysed in cold medium containing protease inhibitors (cOmplete, EDTA-free Protease Inhibitor Cocktail) and the supernatant (900 xg, 15 min) was used for western blotting. Proteins were separated (4%–8% RunBlue SDS gel, Expedeon, San Diego, CA), transferred to a polyvinyl difluoride (PVDF) membrane using an iBLOT gel-transfer system (ThermoFisher), blocked in Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.2% Tween-20 and 5% BSA for 1 hr, washed in the same medium, and incubated with primary antibody (16 hr, 4°C) in the blocking buffer. After washing (3 × 5 min), the membrane was incubated with secondary antibody (1 hr, 20°C), washed (3 × 5 min). Bands were visualized using ECL Prime western blotting detection reagent and a Syngene Pxi chemiluminescence detection system with GeneTools software.

Protein was extracted from monocytes using an extraction reagent for mammalian cells including a protease inhibitor cocktail (cOmplete Lysis-M, pH 7.6, Roche, Denmark). Proteins were separated by 10% sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (10% RunBlue SDS gel, Expedeon, UK) at 120 V for 45 minutes and transferred to polyvinylidendifluoride membranes at 100 V for 75 minutes (Immun-Blot LF PVDF, Bio-Rad, USA). Membranes were blocked with blocking buffer (Superblock, Thermo Fisher Scientific, USA) for 1 hour at room temperature and incubated with the primary antibodies rabbit anti-human NQO1 (ab34173, Abcam, UK) or rabbit anti-human ACTB (sc-130656, Santa Cruz Biotechnology, Germany). After washing with tris(hydroxymethyl)aminomethane-buffered saline, the membranes were incubated with the fluorescence-labeled secondary antibody F(ab′)₂-goat anti-rabbit IgG (H+L) Alexa Fluor 488 (Fisher Scientific, Denmark). Imaging was performed using a Carestream Imager 4000pro (Fisher Scientific, Denmark) at 535 nm emission with an excitation wavelength of 470 nm.