In in vitro treatment, whole cell extracts from intact cells were treat ed with wortmannin during heating. Cell survival assay Cell survival selleck chemicals Vandetanib after heating at 44?C for 0, 15, 30, 60, 90 or 120 min was quantitated by plating cells into 25 cm2 flask containing the medium. Ten to fourteen days later, cell colonies were rinsed with PBS, fixed with methanol, stained with 2% Giemsa solution. Colonies containing at least 50 cells were count ed. The number of cells per colony was determined prior to experiment. Western blotting analysis Detailed procedure of Western blotting is described else where. Aliquots of whole cell extracts were used for Western blotting analysis of Bax and p53. After electrophoresis on 15% polyacryla mide gels containing 0.

1% SDS and electrophoretic transfer onto Poly Screen PVDF membranes, the proteins on each membrane were incubated with the anti human Bax polyclonal antibody Ab 1, anti human p53 monoclonal antibody DO 1, anti human phosphorylated p53 polyclonal antibody Phospho p53 or anti human WAF1 monoclonal antibody EA10. The bands were visualized using horseradish peroxidase conjugated goat anti rabbit or anti mouse IgG anti body and the BLAST Blotting Amplification System. Preparation of nuclear or whole cell extracts for gel mobil ity shift assay Nuclear extracts were prepared from A 172 transformed cells 6 hr after heat treatment, heat and glycerol treat ments or no treatments as in vivo treatment samples. As in vitro treatment samples, whole cell extracts were prepared from intact A 172 transformed cells suspended in extrac tion buffer, 0.

5 mM phenylmethyl sulfanylfluo ride, 25% glycerol, 1. 2M spermidine and were treated with glycerol, heat or combination of glycerol and heat, and subsequently incu bated for 30 min at 37?C. The procedures of nuclear pro tein extraction are described previously. Shortly, the cells were washed with PBS and suspended in washing buffer and then homogenized on ice in hy potonic buffer, and 0. 5 mM phenylmethyl sulfanylfluoride with a hand driv en Dounce homogenizer. The homogenates were centri fuged to precipitate the nuclei, which were resuspended in extraction buffer glycerol, 1. 2M sper midine. The resulting nuclear suspensions were centri fuged to precipitate the chromatin and the nuclear extracts were collected and dialyzed against binding buffer glyc erol. The protein concentration of each extract was quan tified using a BIO RAD Protein Assay Kit with bovine serum albumin as the standard. Gel mobility shift assay The p53 p53CON binding activity was measured by a gel shift assay using a synthetic double stranded DNA frag ment encoding the p53CON on the Drug_discovery upstream of bax gene as a probe. Detailed procedure is described elsewhere.