Cells were rinsed twice with cold PBS, and total protein was extracted with ice-cold lysis buffer (Cell Signaling Technology, Beverly, MA) supplemented with protease inhibitor cocktail (Roche Diagnostics Ltd., Mannheim, Germany), sodium fluoride (10 mM), and phenylmethylsulphonyl fluoride (1 mM) to prevent protein degradation. Cells were scraped and forced through a thin needle two or three times. The lysates were incubated for 20 minutes on ice and centrifuged at 15,000g at 4°C for 20 minutes. Supernatants were transferred into fresh tubes. Protein concentration was measured using a modified Lowry protein assay kit (Thermo Scientific, Rockford, IL).
Equal aliquots (10 μg) of protein were separated using 12% SDS-PAGE and transferred to polyvinylidene fluoride membrane for 1.5 hours at 4°C, using a Bio-Rad (Hercules, CA) miniprotein-III wet transfer unit. The transfer membranes were then incubated with blocking solution (5% nonfat dried milk dissolved in Tris-buffered saline–Tween buffer [pH 7.6], 10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20) for 1 hour at room temperature. The membranes were incubated with a rabbit polyclonal antibody against copper/zinc superoxide dismutase (Cu/ZnSOD, 1:2000), manganese SOD (MnSOD, 1:2000), extracellular SOD (ecSOD, 1:2000) (Stressgen Biotechnologies, Victoria, BC), or α-tubulin (1:10,000; Sigma-Aldrich) at 4°C overnight. This was followed by incubation of the membranes with horseradish peroxidase–conjugated anti-rabbit IgG (1:2000; Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 2 hours. Specific antibody–antigen complex was detected using an enhanced chemiluminescence Western blot detection system (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The amount of detected protein was quantified by Photo-Print Plus software (ETS Vilber-Lourmat, Marne-la-Vallée, France).