which serves as the hydride donor to PA 824 in the Rv3547 catalyzed r

Pictures were taken using a digital camera to record the prior to colony counting as described previously. H460 and H157 cells were maintained in RPMI 1640 with 10% fetal bovine serum. A549 cells were maintained in F 12K medium with 10% fetal bovine serum. The rapamycin resistant A549 cell line was established as described previously. Crizotinib Briefly, A549 RR was established by exposing the rapamycin sensitive A549 parental cells to gradually increasing concentrations of rapamycin from the initial 1 nM to the final 20 uM over a 6 month period as described. Preparation of cell lysate and Western blot Cells were washed with cold PBS and resuspended in ice cold EBC buffer containing protease inhibitor mixture set I. Following cell lysis by sonication and centrifugation at 14,000 g for 15 min at 4 C, the resulting supernatant was collected as the total cell lysate. As previously described, Western blot was performed by loading 50ug of protein per lane on an 8?12% SDS PAGE, followed by protein transfer to nitrocellulose membrane for analysis of specific protein. RNA interference, plasmids and transfection Human Akt shRNA plasmid is a target specific lentiviral vector plasmid Immune system encoding a 19 25 nt shRNA designed to knock down gene expression. The control shRNA plasmid A encodes a scrambled shRNA sequence that will not lead to the specific degradation of any cellular message. Both Akt shRNA and control shRNA plasmids were purchased from Santa Cruz Biotechnology. Bad cDNA or HA tagged ubiquitin plasmid was performed using NanoJuice transfection Kit according to the manufacturers instructions. Subcellular fractionation was performed as previously described. Colony formation Oprozomib assay A549 P and A549 RR cells were trypsinized and plated in 6 wellplate, then treated with rapamycin. Every 3 days, the medium was replaced with fresh medium containing the rapamycin. After 10 days of treatment, the medium was removed and cell colonies were stained with crystal violet. Important regulators of this complex pathway are the proteins of the Bcl 2 family. Their main function is to control the release of apoptotic proteins from the mitochondria. Members of the Bcl 2 family interact with a variety of proteins and therefore accelerate the rupture of the outer membrane or the mitochondria, which leads to a release of pro apoptotic proteins and the triggering of apoptosis. Moreover, compounds 6 and 7 will be obviated from the following analyses, because of the great number of hydrogen donors, which do not comply with the Lipinski Rule of five. To make a prediction of the binding affinity for the remaining four compounds from the in computer assisted screening, the molecules were docked into the binding groove of the antiapoptotic protein Bcl XL.