Proteins were normalized to the number of total cells counted in each culture at the time of super natant collection. Western blot analysis Rabbit polyclonal antibodies against AKT and phospho AKT were purchased from Cell Signaling Tech nology, Inc. Rabbit polyclonal antibodies against I��B. NFB2p52 and RelA, as well as mouse monoclonal antibody against lamin AC were purchased from Santa Cruz Regorafenib chemical structure Bio technology, Inc. mAb against actin was obtained from Sigma Aldrich. Whole cell extracts and nuclear extracts were prepared as described previously. Twenty five ug or 10 ug of proteins per sample were run on a 10% SDS polyacrilamide gels, transferred to nitrocellulose membranes and blocked with 5% non fat milk in Tris buffered saline supplemented with 0. 1% Tween 20 for 1 h at room temperature.
The mem branes were then incubated in the same solution over night at 4 C with primary antibodies at the following dilutions anti AKT 11000. anti phospho AKT 1500. anti I��B 1200. anti NFB2p52 1200. anti RelA 1500. anti lamin Inhibitors,Modulators,Libraries AC 1500, anti actin 11000. The latter two antibodies were used as internal standards for loading. Immunodetection was carried out using appropriate horseradish peroxidase linked secondary antibodies and enhanced chemiluminescence detection reagents. Where indicated, films were scanned on a GS 710 Calibrated Inhibitors,Modulators,Libraries Imaging Densitometer and analyzed by means of Quantity One Software Version 4. 1. 1. Transient transfection with small interfering RNA Inhibitors,Modulators,Libraries targeting RelA Oligonucleotide siRNA targeting RelA to be used as a control were purchased from Sigma Proligo.
For Western blot analysis, HCT1163 6 and M10 cells were suspended in culture medium without antibiotics, seeded Inhibitors,Modulators,Libraries into 60 mm dishes and allowed to adhere at 37 C for 18 h. The cells were then transfected with 100 nM siNFp65 or scrNFp65 using Oligofecta mine Reagent according to the manufacturers protocol. As an additional control group, the cells were treated with the transfection re agent alone. After 72 h of cul ture at 37 C, the cells were recovered, plated Inhibitors,Modulators,Libraries and subjected to a second transfection as described above. Total cell extracts were prepared 7 days after the second transfection. For chemosensitivity assays, HCT1163 6 and M10 cells recovered after the first transfection were seeded into 96 well plates, allowed to adhere at 37 C for 18 h, and then subjected to the second transfec tion.
After 24 h of incubation, the cells were exposed to graded concentrations of TMZ selleck chemicals plus BG or to BG alone. The plates were incubated at 37 C for 6 days and cell proliferation was then evaluated by the MTT assay, as previously described. Four replica wells were used for each group. TMZ concentration producing 50% inhibition of cell growth, was calculated on the regression line in which absorbance values at 595 nm were plotted against the logarithm of drug concentration.