5% crystal violet and colonies with more than 50 cells were counted. Clonogenic survival curves were fitted using the linear Vandetanib cancer quadratic model and the surviving frac tion Inhibitors,Modulators,Libraries after 4 Gy was calculated using the Inhibitors,Modulators,Libraries and B values obtained from the curve. Kinase inhibition Clonogenic cell survival assays western blot analyses For clonogenic cell survival assays, cells were incubated with the kinase inhibitor for 16 h and then irradiated with 4 Gy. Thereafter, cells Inhibitors,Modulators,Libraries were treated with the kinase inhibitor for 72 h and subse quently cells were incubated in drug free medium. After 1. 5 3 weeks, cells were stained with crystal violet and colonies were counted. Survival fraction after combined treatment with 4 Gy and the kinase inhibitor was calcu lated by correcting for plating efficiency of the untreated control or by correcting for plating efficiency of cells treated with the inhibitor alone.

For western blot analyses, cells were treated with the inhibitor for 16 h followed by irradiation with 4 Gy and harvested 4 h after radiotherapy or 20 h after kinase treatment. Cells were lysed in RIPA buffer and protein was quantitated using a standard Bradford absorbance assay. Proteins were separated by SDS PAGE Inhibitors,Modulators,Libraries and blotted onto PVDF membrane. Membranes were incubated with the appropriate primary antibodies followed by incubation with HRP conjugated antibodies. Finally, proteins were detected using chemilumines cence. Antibodies against the following antigens and HRP conjugated goat anti rabbit IgG were purchased from Cell Signaling Technology, HRP conjugated goat anti mouse IgG was purchased from Santa Cruz Bio technology, and tubulin was obtained from Calbiochem.

Statistics Correlations between expression levels of phospho kinases and SF4 values were assessed using the Spearman correlation test. To determine additive effects of combined treatment, differences between survival after 4 Gy and 4 Gy inhibi tor were tested for significance using the Inhibitors,Modulators,Libraries Mann Whitney test. To determine supra additive effects of combined treatment, differences between survival after 4 Gy and 4 Gy inhibitor corrected for effect of inhibitor alone were tested for significance using the Mann Whitney test. Tests were performed using Prism was calculated for each cell line. To determine which kinases are important for cell www.selleckchem.com/products/carfilzomib-pr-171.html survival after radiotherapy in HNSCC, we quantified the expression of a panel of phospho kinases using an antibody based array in untreated and irradiated cells. The effect of radiotherapy on most phospho kinases varied widely among cell lines, only the ex pression of p Chk2 was increased in all cell lines after radiotherapy. The expression levels of mul tiple phospho kinases were found to be significantly cor related with radiosensitivity.