The cells were cotransfected with AP 2and or Ku80 expression vectors or the corresponding empty vectors. The luciferase activity in the cells transfected with the empty expression vector was www.selleckchem.com/products/ABT-263.html considered as equal to one. As previ ously shown, AP 2stimulated the activity of the reporter containing the wild type AP2BS. How ever, the activation was reduced in 70 32 cells expressing less Ku80. Ku80 expression vector alone had no effect on the promoter activity. However, co transfection of Ku80 with AP 2expression vectors induced a significant increase in the promoter activity in both cell lines. Interestingly, transfection of Ku80 restored the activation capacity of AP 2in 70 32 cell line. Figure 4C presents the AP 2and Ku80 protein levels in the transfected cells.

To complete this investigation, the reporter vectors activities were measured in BT 474 and SKBR3 cells 72 hours after transfection of Ku and AP 2 siRNAs. The Luciferase activity in cells trans fected with the negative siRNA Inhibitors,Modulators,Libraries was considered as equal to one in each condition. As expected, downregulation of AP 2and AP 2inhibited only the activity of the reporter containing the functional AP2BS. Ku70 or Ku80 siR NAs also inhibited significantly the activity of this reporter vec tor. Furthermore, when Ku70 or Ku80 siRNAs were co transfected with the AP 2siRNAs, the activity of p86 AP2BS Luc was further reduced. In contrast, AP 2?, Ku70 and Ku80 siRNAs did not modify significantly the activity of the vector containing the mutated AP2BS. However, co transfection of AP 2with Inhibitors,Modulators,Libraries Ku70 or Ku80 signif icantly reduced the activity of the reporter containing the mutant AP2BS.

Together, these results suggest that Ku proteins regulate ERBB2 gene expression through the proximal promoter, by an AP 2 dependent mechanism. Ku70 80 proteins are recruited to the ERBB2 proximal Inhibitors,Modulators,Libraries promoter Next, we investigated Ku binding to ERBB2 gene promoter by chromatin immunoprecipitation. Inhibitors,Modulators,Libraries Cross linked chroma tin was extracted from BT 474 and SKBR3 cells. We also extracted chromatin from the same cell type transfected with Ku or AP 2siRNAs. Chromatin fragments were immunoprecipitated with antibod Inhibitors,Modulators,Libraries ies recognizing AP 2and AP 2?, Ku70, Ku80 and RNA Polymerase II. The Pol II antibody was used as a positive control for the recruitment of the transcriptional machinery on the ERBB2 promoter. Three regions of the ERBB2 promoter were amplified.

mean The 500 bp region contains a high affinity AP2BS. The 100 bp region contains the CAAT and TATA boxes and corresponds to the ERBB2 core promoter. The 6900 bp sequence was used as an AP2BS negative control. The Ku binding sequence from the Glucokinase gene promoter was used as a positive control for Ku proteins binding. Exper iments were repeated three times and the average quantities of DNA were reported to the No antibody condition.