The clonies were picked, grown, and then plasmids were extracted, screened and analyzed by agarose gel electrophoresis, and one named AdEasy-GFP-NDRG2 selected. The construction of
recombinant adenovirus AdEasy-GFP-NDRG2 was performed as described by Tran et al . Infectious viruses were purified by plaques. All recombinant adenoviruses were amplified on human embryonic kidney cell line 293 and purified by double cesium chloride density gradient ultracentrifugation. C646 Titers of the adenoviral stocks were determined by plaque assay on 293 cells. Photograph of viral plaque formation to count viral titer (plaque assay). HEK-293 cells, which grew confluently on the bottom of the 24-well plastic plate (1.5 cm diameter each), were infected with serially diluted solutions containing adenoviral virus, and then cultured over night to make viral plaque. The number of plaques indicates the number of the infectious virus (= viral titer, as plaque forming unit). AdEasy-GFP-p53
was provided by Dr. Lintao Jia. Cell Culture The human renal clear-cell carcinoma lines A-498 and the human embryonic kidney cell lines HEK-293 were obtained from the American Type Culture Collection (ATCC) and maintained as recommended. A-498 was cultured in Minimum Essential Medium (MEM) with 2 mM L-glutamine and Earle’s BSS adjusted to contain 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium selleck chemical pyruvate. HEK-293 was cultured with Dulbecco’s Modified Eagles’ Medium (DMEM). All the culture fluid was supplemented with 10% fetal calf
serum (FCS) and all cells were Urocanase cultured with 5% CO2 at 37°C in a humidified chamber. Western blot analysis Cells were washed with ice-cold PBS and lysed in a RIPA buffer [50 mM Tris (pH7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS] containing PMSF (1 mM) and protease learn more inhibitors (2 μg/ml; Protease Inhibitor Cocktail Set III, Calbiochem) on ice for 30 minutes. The lysates were clarified by centrifugation at 13,000 × g for 30 minutes at 4°C. The total protein concentration was estimated using Protein Assay Kit (Bio-Rad, Richmond, CA). 30-80 μg protein samples were loaded on a 12% SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes. After being blocked with TBST [20 mM Tris (pH7.5), 150 mM NaCl, 0.01% Tween-20] containing 5% non-fat dry milk for 1 hour at room temperature, membranes were probed with an appropriate antibody overnight at 4°C followed by a horseradish peroxidase (HRP)-linked goat anti-mouse or anti-rabbit antibodies at room temperature for 1 hour. The membranes were analyzed using super ECL detection reagent (Applygen, Beijing, China).