Un scan it gel analysis software

Cells were treated with different concentrations of juglone (0, 20, and 40 μM) for 48 h. Total protein extracts were obtained from lysis buffer (150 mM NaCL, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris-Cl pH 8.0, 2 ug/mL aprotinin, 2 ug/mL leupeptin, 40 mg/mL of phenylmethylsulfonyl fluoride, 2 mM DTT). The protein concentration was determined by the Bradford assay (BioRad, Hercules, CA), and samples were separated on SDS-PAGE, and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were immunoblotted with primary Abs against cleaved caspase 9 (Cell Signaling Technology, 1:1000), P-p38 (Cell Signaling Technology,1:1000), and β-actin (Cell Signaling Technology,1:10000) overnight at 4 °C, followed by horseradish peroxidase (HRP) conjugated secondary Ab (BioRad,1:3000). Detection was carried out using Supersignal West Femto Chemiluminescent Substrate (Pierce, Rockford, IL). β-actin was taken as reference and the band intensities were quantified using UN-SCAN-IT gel analysis software (Silk Scientific, Orem, UT).

GG, SG, ADG, and PDG were removed from euthanized mice and homogenized through mechanical disruption and sonication. Total protein was isolated through previously described methods[25 (link)]. A large-format Hoefer SE 600 series sodium doedecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) system was used to separate 400 ng of protein samples by molecular weight. Samples were run at 8°C for 29–32 hours though a 6% acrylamide, 30% glycerol separation gel and a 4% acrylamide, 30% glycerol stacking gel[26 (link), 27 (link)]. MyHC isoforms were detected though silver staining and identified thorough relative mobility as previously described[28 (link)]. Optical density of each band was digitally assessed thorough Un-Scan-It Gel Analysis Software (Silk Scientific, Inc.) and was used to determine MyHC isoform composition of each muscle, calculated as a relative percentage of total MyHC. Proteins from soleus and extensor digitorum longus (EDL) muscles were used as control samples to verify the relative positions of MyHC I and MyHC 2b, respectively, as previously described[28 (link)].

Changes in proteins over time during incubation with pepsin, trypsin, and chymotrypsin were visualized with reducing SDS-PAGE. Laemmli sample buffer and 10× tris/glycine/SDS running buffer were obtained from Bio-Rad (Hercules, CA), β-mercaptoethanol was obtained from Sigma-Aldrich, and SeeBlue Plus2 protein standard was obtained from Invitrogen (Carlsbad, CA). Sample and sample buffer (containing 1% β-mercaptoethanol) were combined in a 1:1 ratio and the mixture was heated for 5 min at 85°C. Samples were run on a 15% tris-HCl Ready Gel (Bio-Rad Laboratories) at constant voltage (200 V). Gels were stained with Imperial Protein Stain (Thermo Fisher Scientific, Rockford, IL). Band density was quantified with UN-SCAN-IT gel analysis software (Silk Scientific, Inc., Orem, UT).