Quantity one 1d image analysis software

All data are provided as a mean ± standard deviation (SD). The analysis of variance test (ANOVA) and Student–Newman–Keuls test (SNK) for multiple comparisons were applied for the data analysis among groups while a paired Student’s t-test was used for the data analysis within the same group, considering p-values < 0.05 as statistically significant.
QuantityOne 1D image analysis software (version 4.6.7, Bio-Rad) and the PDQuest 2D image analysis software program (version 7.3.1, Bio-Rad), were used to evaluate the protein bands revealed by 1-DE and the protein spots detected by the 2-DE analysis, respectively. The signal intensity of the protein bands and spots were expressed as optical density (OD). In order to correct the variability due to the staining procedure, band volumes were normalized as percentage of the total OD of all the bands detected in the gel while 2D gel images were normalized by a filtration process. Protein spots with an expression variance > 1.5-fold were selected as significantly different between the studied groups.

Mouse liver tissues were lysed with radioimmunoprecipitation (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 50 mM Tris-HCl (pH 7.5)) supplemented with protease inhibitor cocktail. Equal concentrations of proteins were diluted in the SDS sample buffer (50 mM Tris-Cl at pH 6.8, 2% SDS, 100 mM DL-dithiothreitol (DTT), 10% glycerol), separated on an 8–12% polyacrylamide gel, and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking the membrane with 5% skim milk for 30 min, the target antigen was reacted with the primary antibody at RT for 2 h. The membrane was then incubated with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody at RT for 1 h. The immunoreactive bands were detected using an enhanced chemiluminescence system (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific). The band intensity was measured using Quantity One 1D image analysis software (Bio-Rad).

Rac-GTP levels in cell lines and tumors were assessed by pull-down assay with a GST fusion protein containing the Rac1 binding domain of PAK1 (GST-PBD) as described previously [14 (link)]. Briefly, MCF7 cell lines were seeded in six well plates, serum starved for 48h and then stimulated with EGF (100 ng/ml) for 5 min or HRG (10 ng/ml) for 10 min. After stimulation cells were lysed in 400 μl of lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.5% Igepal, 5 mM β-glycerophosphate, 1 mM DTT, 1 mM Na₃VO₄, 50 mM NaF, protease inhibitors (Cømplete, Roche Diagnostics), and 10 μg/sample of GST-PBD. Tumors were homogenized in the same lysis buffer (1:10 w/v) without GST-PBD, precleared by centrifugation at 14,000 rpm for 10 min at 4°C and then, 10 μg of GST-PBD was added to 1 ml of supernatant. Tumor and cell lysates were incubated with glutathione-Sepharose beads (GE Healthcare) for 1 h at 4°C. After incubation, samples were extensively washed, boiled in SDS-PAGE sample buffer and separated by electrophoresis. Bound Rac1 proteins were detected by immunoblotting using anti-Rac antibody (610651, BD transduction laboratories). Rac-GTP levels were quantitated using the Quantity One 1D image analysis software (Bio-Rad).

C2C12 myotubes and mouse gastrocnemius muscles were lyzed with RIPA buffer [150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 50 mM Tris-Hcl (pH7.5)] supplemented with a protease inhibitor cocktail. Equal concentrations of proteins were diluted in SDS sample buffer (50 mM Tris-Cl at pH 6.8, 2% SDS, 100 mM DL-dithiothreitol (DTT), 10% glycerol), separated on 8–12% polyacrylamide, and transferred to a poly (vinylidene fluoride) (PVDF) membrane sheet. After blocking the membrane in 5% skim milk for 30 min, the target antigen was reacted with the primary antibody at RT for 2 h. The membrane was then incubated with the secondary antibody (horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibodies) at RT for 1 h, after which an immunoreactive band was detected using an enhanced chemiluminescence system (Pierce ECL Western Blotting Substrate; Thermo, Rockford, IL, United States). Band intensity was measured using Quantity One 1D image analysis software (Bio-Rad, Hercules, CA, United States).

After treatment with OxLDL and cyclophilin A, protein lysates were prepared and separated on SDS-PAGE and resolved proteins were transferred on to nitrocellulose membrane. The membrane was incubated with 1:1000 dilutions of each of the following primary antibodies at 4 °C for 18 h: Mouse anti-cyclophilin A (ab-58144), Mouse anti β Actin (sc-47778), rabbit anti-CD 36 (#14347), Rabbit anti-LOX-1 (ab174316) as per manufacturer’s instructions. The membrane was then incubated with specific secondary antibodies: anti mouse IgG HRP (ab-6789) and anti rabbit IgG-HRP (ab-97051) at a dilution of 1:5000. The proteins were visualized with clarity western ECL substrate. The bands were analyzed by Quantity One 1D image analysis software (Bio-Rad, USA).