Imagequant tl software package

The levels of total protein bound HNE were determined in the Free Radical Biology in Cancer Shared Resource Facility (FRBC SRF) at the University of Kentucky. The mitochondrial samples (pellets) were thawed and resuspended in a small volume (75–150 uL) of ice-cold homogenization buffer (0.32 M Sucrose, 10 mM HEPES pH 7.4, 2 mM EDTA, protease inhibitors). Five microliters of the homogenized sample were mixed and diluted with an equal volume of 12% SDS. Samples were further denatured with 10 µl of modified Laemmli buffer (0.125 M Trizma base, 4% SDS and 20% Glycerol) for 20 min at room temperature. Next, 250 ng of the derivatized protein were loaded in each slot (48-well slot-format Bio-Dot SF Apparatus with nitrocellulose membranes, pore size 0.2 µm, Bio-Rad, Hercules, CA). The antibody reaction was developed by using BCIP (5-bromo-4-chloro-3-indolyl-phosphate) in conjunction with NBT (nitro blue tetrazolium). The immunodetection was performed by using 1:5000 Anti-4 hydroxynonenal antiserum (Alpha Diagnostic International Inc., San Antonio, TX) and goat 1:7500 anti-rabbit IgG (Sigma-Aldrich, St Louis, MO) antibody for the secondary detection. The nitrocellulose membranes were scanned by Photo Scanner (Epson Perfection V600, Long Beach, CA), and slot blot line densities were quantified by the ImageQuant TL software package (GE Healthcare Bio-Sciences, Piscataway, NJ).

The steady-state level of each Ntg1-TAP fusion protein variant was assessed by immunoblotting whole cell lysates with the rabbit polyclonal anti-TAP antibody (1:3,333 dilution, Open Biosystems) to determine the relative level of differentially modified Ntg1 products. An anti-3-phosphoglycerate (PGK) antibody (1:10,000 dilution; Invitrogen) was used as a control determine the relative level of protein lysate loaded into each lane.
The analysis of immunoblots was performed utilizing the ECL Plex immunoblotting detection system (Amersham), the Typhoon Trio variable mode imager (GE Healthcare), and the ImageQuant TL software package (GE Healthcare). To quantify the percentage of modified Ntg1-TAP, the ratio of modified Ntg1 bands to total Ntg1 signal (including modified and unmodified) was determined for wildtype Ntg1 and each lysine to arginine amino acid substitution variant of Ntg1. Previous work demonstrates that modified Ntg1 contains at least one covalently linked SUMO and the size of higher bands is consistent with multiple SUMO additions(24 (link)). Standard error of the mean was calculated for each. The two-sample Student’s t-test was employed to test for significance (α=0.05).