C digit chemiluminescent western blot scanner

Protein isolation was performed as follows. Isolated heart tissues from rats of all groups were washed with ice-cold PBS followed by minced and homogenization in cold protein lysis buffer and protease inhibitor cocktail (Ansari et al., 2013 ). The cell lysates were incubate on ice for 60 min with vortex mixing after every 10 min, followed by centrifugation for 10 min (12,000 RPM, 4 °C), to obtained total cellular proteins. Total protein content was measured according to the well-established method of Lowry et al. (1951) (link). Western blot analysis was done by following the previously described method of Ansari et al. (2013) . Briefly, protein (25–50 µg) from each group was separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoretically moved to nitrocellulose membranes. Protein blots was incubated overnight at 4 °C with primary antibodies against GST, NF-κB p65, pNF-κB p65 and NQO1 and peroxidase-conjugated secondary antibodies at 25 °C. Bands were visualized using the enhanced chemiluminescence method (GE Health Care, Mississauga, Canada). Band intensities were calculated comparative to β-actin bands using an image analysis system (ImageJ® image processing program, National Institutes of Health, Bethesda, USA). Images were capture by using C-Digit chemiluminescent western blot scanner (LI-COR, Lincoln, USA).