Un scan it gel version 6

The protein concentration of each TA muscle was optimized so that 0.4 μg of protein was added to each gel channel in duplicate. SDS-PAGE was performed on each concentrate with a 0.75 mm thick 6% acrylamide/30% glycerol separating gel (18 × 16 cm) and a 4% acrylamide/30% glycerol stacking gel. A silver staining kit (SilverQuest Silver Staining Kit, Life Technologies, New York, USA) was used to stain the gel for visualization of protein bands. Each silver-stained gel was imaged digitally. An investigator masked to rat age and treatment determined the optical density of each band using computer-assisted image analysis and densiometry (UN-Scan-IT gel Version 6.1, Silk Scientific, Inc, Utah, USA). The ratio of the density of an individual MHC isoform band to the total density within a column was used to determine the percentage of each MHC isoform per lane ^{19 ,44 (link),46 (link)-51 (link)}. Good to excellent reliability using these procedures has been demonstrated in previous studies ^{50 (link)}.

All data columns are shown as mean ± S.E.M. In Western blots, n is presented as the number of separate experiments (minimum of 3). Western blots were quantified using Un-Scan-It gel version 6.1 (Silk Scientific Inc., Orem, UT). Statistical differences between control and experimental conditions were determined by using Mann Whitney non-parametric test followed by Dunnet’s post hoc test or a Mann & Whitney non-parametric test when comparing only two conditions within R Software (R-project). P values < 0.05 were judged to be statistically significant. Graphs were generated using Graphpad Prism 7 software.

Hepatic cell lysates (40 μg proteins) were separated by 12% SDS-PAGE and subjected to immunoblot analyses, as previously described [31 (link)]. Specific antibodies to CYP2E1, iNOS, 3-NT and β-actin, as a loading control, were then incubated overnight at 4°C. After removal of the primary antibodies and followed by three separate steps of washing, the nitrocellulose membranes were incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase. Protein bands were detected by enhanced chemiluminescence and their densities quantified using UN-SCAN-IT gel version 6.1 from Silk Scientific (Orem, Utah 84059 USA). For the immunoprecipitation experiment, 400 μg of hepatic cell lysates from each mouse was used to prepare a total of 2 mg proteins/sample (pooled from 5 mice/sample out of 10 mice/strain to prepare 2 samples/strain) and the specific antibody (5 μg) to collagen 1A1 or HSP90 was used with Pierce^™ protein A/G magnetic beads from Life Technologies (Grand Island, NY, USA) following the manufacturer’s protocol. The immunoprecipitated proteins were subsequently analyzed by immunoblot analysis using the specific antibody against collagen 1A1, HSP90, or 3-NT (Table 2).