Horseradish peroxidase conjugated anti rabbit secondary antibody

The cytotoxicity of E1h was determined by 3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay as described [40 (link)]. Cells (10,000 per well) cultured for 1 day in 46-well plates were replaced with medium containing 100 nM E1h after washed with PBS, and further cultured. The indicated time point later, the surviving cells were stained with MTT and quantified by absorbance at 540 nm. The MTT assay results were plotted with mean ± S.D. of three experiments.
The in vivo stability of E1h was measured by western blotting against E1h in tumor tissue obtained from PC3-bearing mice after in vivo optical imaging. The tumor tissues dissected from mice were soaked in PBS buffer containing 1X protease inhibitor cocktail solution (GenDEPOT) and grinded with homogenizer. The tumor samples were centrifuged in 12,000 rpm for 10 min. The supernatants (100 μg protein) were mixed with SDS sample buffer and separated with 12% SDS-PAGE. After transferred to nitrocellulose membranes, proteins were first probed using a mouse anti-his tag antibody (1:1000 dilution, Santa Cruz Biotechnology, CA) and then a horseradish peroxidase-conjugated anti rabbit secondary antibody (1:2000 dilution, Amersham, UK). As a loading control, beta-actin was detected using the same (stripped) membrane.

Frozen liver tissues of each mouse were pulverized in liquid nitrogen, and lysed with PRO-PREP^TM Protein Extraction Solution (iNtRon BIOTECHNOLGY, Gyeonggi-do, Korea) containing protease inhibitors for 20 min on ice. The lysates were centrifuged at 1,300 rpm (20 min, 4°C), and the total protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA, USA). Protein samples were separated with 10% SDS-PAGE and transferred to nitrocellulose membranes (Whatman, Maidstone, Kent, UK). The membranes were blocked with 5% skim milk in Tris-buffered saline solution containing Tween-20 (Sigma-Aldrich), and then incubated overnight at 4°C with primary antibodies to PPARγ (1:1000; Santa Cruz sc-7196, CA, USA) and monoclonal mouse anti-β-actin (1:2500; Sigma-Aldrich A2228). Membranes were washed with Tris-buffered saline containing 0.05% Tween-20 and incubated with horseradish peroxidase–conjugated anti-rabbit secondary antibody (1:5000; Amersham Pharmacia Biotech NA934, Piscataway, NJ, USA). Protein bands were visualized using an enhanced chemiluminescence system (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The band densities were quantified by Multi Gauge V3.0 program (Fujifilm Life Science), and normalized to the protein expression levels of β-actin.