As depicted in Figure 1B, the IC50 for FLLL32 ranged from 0. 75 1. 45 uM for the

After 48 h or 72 h, cells were harvested and ana lyzed for annexin V binding by flow cytometry. In control cells, the irrelevant control siRNA was used. All experiments were performed in triplicate. Preparation of subcellular ARN-509 fractions Cells were resuspended in cell lysis buffer containing 20 mM Hepes pH 7. 4, 1 mM MgCl2, 10 mM KCl, 0. 3% NP40, 0. 5 mM DTT, 0. 1 mM EDTA and pro tease inhibitors, and placed at 4 C for 5 min. The lysates were centrifuged at 14000 g for 5 min at 4 C, and the supernatant contain ing the cytoplasmic fraction was stored in aliquots at 80 C. The pellets were resuspended in cell lysis buffer adjusted to 20% glycerol and 0. 35 M NaCl and placed at 4 C for 30 min. After centrifugation at 14000 g for 5 min at 4 C, the supernatant, containing the nuclear proteins, was stored at 80 C.<br><br> Protein amounts were determined before use with the micro BCA protein determination AT7519 価格 kit. where R is either H or biotin. The synthesis of decoy oligonucleotides with R H has been published elsewhere. For biotin addi tion, 7 10 nanomoles of the oligodeoxynucleotide bear ing 3 and 5 aminoalkyl linkers were dissolved in 20 uL of 0. 1 M NaHCO3. EZ Link NHS biotin was added, and the mixture was incubated at room temperature for 6 16 h in the dark. Then 25 uL of water were added, and the modified oligodeoxynucleo tide was separated from the excess of hydrolyzed reagent by two consecutive separations on Micro Bio Spin 6 columns following the manufacturers recom mendations. After the second spin, the biotinylated oli godeoxynucleotide was precipitated with ethanol sodium acetate.<br><br> In control experiments the previously オーダー Alisertib published decoy NF B ODN was used. In some cases FITC labeled or biotinylated decoy ODNs were obtained from Sigma Aldrich. Note that the oligonucleotides used for cell death induction, pull down assays and whole cell pull down assays were similar and could be used inter changeably, except that for pull down biotinylated oligo nucleotides had to be used. Preparation of liposomes Liposomes were formulated using a cationic lipid carbamoyl] choles terol iodide and neutral colipid dioleoyl phosphatidylethanolamine, as previously described. The concentration of cationic lipid was monitored by UV spectroscopy at 226 nm and the value was used to calculate the charge ratio assuming one positive charge for each cationic lipid molecule.<br><br> Gel electrophoresis, western blotting Cells were washed in PBS, lysed in sample buffer, 2% sodium dodecyl sulfate, 20% glycerol, 1 mM sodium vanadate, 1 mM dithiothreitritol and 0. 01% bromophenol blue, sonicated and stored at 70 C. Proteins were separated on SDS PAGE and transferred onto nitrocellulose membranes, mem branes blocked with 5% dry skimmed milk in TBS were incubated with antibody overnight at 4 C. Anti phos photyrosine 705 STAT3, anti STAT3, anti NF B p50, anti NF B p65, anti STAT1, and anti OCT1 were from Cell Signaling, anti karyopherin importin a was from Santa Cruz. Blots were washed in TBS T, incu bated with peroxidase coupled goat anti mouse or goat anti rabbit secondary antibody washed in TBS T and revealed by chemiluminescence and autoradiography.