Science lab 2001 image gauge 4

Cell lysates were prepared following treatment of A549 cells with SHK as described previously [36 (link), 38 (link)] and used for immunoblotting analysis. An equal amount of cytosolic protein (20 μg) from each sample was then electrophoretically separated by 12% sodium docecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For the analysis of cytochrome c, mitochondrial and cytosolic fractions were prepared from the cells and the cells and the proteins (50 μg) were separated from 15% SDS-PAGE. After being transferred to polyvinylidene difluoride (PVDF) membranes and blocking the nonspecific binding sites, the membranes were incubated with the primary antibodies (1 : 1000 dilutions) at 4°C overnight. After being washed three times with PBS buffer containing 0.1% Tween 20, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (1 : 5000 dilutions) and visualized by enhanced chemiluminescence (ECL) Plus (Pierce, Rockford, IL, USA). Each membrane was stripped and reprobed with anti-α-tubulin antibody to ensure equal protein loading. The density of each protein band was scanned using Science Lab 2001 Image Gauge 4.0 Software (Fujifilm, Tokyo, Japan) and compared between groups by densitometry (α-tubulin as a reference).

To detect cellular response to the stimulation of t-BHP in aspects of apoptotic signaling mediators (Bax, Bcl-2, and cytochrome c), inflammatory and necrotic regulators (HSP60 and NF-kB), and cell cycle regulated proteins (p53, p21) with immunoblotting, aortic tissues or pelleted cells were lysed on ice with a lysis buffer and were centrifuged at 10,000 x g at 4 ^oC for 10 min. The supernatant was acquired and the protein concentration was determined. For the analysis of cytochrome c (cyto c), mitochondrial and cytosolic fractions were prepared as described previously 38 . The density of each protein band was scanned using ScienceLab 2001 ImageGauge 4.0 Software (Fujifilm, Tokyo, Japan) and compared between groups by densitometry (Actin as a reference).

Whole-cell protein lysates were prepared from cell line monolayers according to standard protocols [23 (link)]. Protein concentrations were determined with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Munich, Germany). Proteins were separated by SDS/PAGE as described by Laemmli and colleagues [24 (link)] and transferred to Hybond-P membranes (GE Healthcare, Little Chalfont, UK). Changes in protein expression and phosphorylation as visualized by chemiluminescence (ECL chemi-luminescent reagent, GE Healthcare) were captured and quantified using a FUJI LAS3000 system with Science Lab 2001 ImageGauge 4.0 software (Fujifilm Medical Systems, Stamford, CT).