Chromatin immunoprecipitation assay The manufacturers protocol fo

Chromatin immunoprecipitation assay The manufacturers protocol for the chromatin immu noprecipitation Assay Kit was followed. Briefly, MCF7 cells or MDA MB 231 cells transfected with control pcDNA vector or CEBP Sorafenib Tosylate b2 and were incubated with 1% formaldehyde for 20 minutes at 37 C. Cells were col lected, lysed, sonicated, and incubated with 4 ug of anti bodies to CEBP a, CEBP b, GATA 1, Stat3, or b actin overnight. PCR was used to amplify DNA bound to the immunoprecipitated histones after reversing the histone DNA cross links. The following primers were used for PCR 472F and 344R. Transfection of small interfering RNA oligonucleotides Small interfering RNA for Stat3, Src, and Con trol were obtained from Dharmacon. Oligonucleotides were transfected using Oli gofectamine following the manufacturers protocol.

For luciferase assay experi ments, MDA MB 468 cells were plated at 4 104 cells per well in a 24 well tissue culture dish. Inhibitors,Modulators,Libraries siRNA were transfected in complete med ium without antibiotics. The 472 Jab1 Luc construct and pRL were cotransfected 24 hours later using Inhibitors,Modulators,Libraries the manufacturers protocol for Lipofectamine PLUS trans fection reagent. Inhibitors,Modulators,Libraries Luciferase assays were performed after 48 hours. Results Mapping the transcription initiation start site of the human Jab1 gene To determine the transcription start site of the Jab1 gene, primer extension analysis was performed. An anti sense oligonucleotide was synthesized corre sponding to the sequence located at the ATG translational start site according to the published sequence.

The P1 primer was radio labeled and extended Inhibitors,Modulators,Libraries using avian mye loblastosis virus reverse transcriptase and analyzed on a polyacrylamide urea gel along with a DNA cycle sequen cing reaction using the same primer. The primer exten sion experiment revealed an extension product of 71 nucleotides using the P1 primer. The nucleo tide 71 bp upstream of the ATG translation start site was designated the 1 transcription initiation site in the numbering of the nucleotide sequence throughout this study. Identification of the transcription elements located in the Jab1 promoter Based on the determined location of the transcription start site, we analyzed the Jab1 5 flanking region for a functional promoter. Using the MatInspector program, which uses TRANSFAC transcription factor binding site matrices, we identified a number of putative transcrip tion factor binding sites upstream of the Jab1 transcrip tion start site.

A typical TATA box was found 40 bp upstream of the transcription start site, along with Inhibitors,Modulators,Libraries a CAAT box at 99 bp. A number of putative transcription factor binding elements were present in the promoter thenthereby sequence, including STAT, ELK, p53, E2F, CEBP, GATA, c Myb, and AP 1. Identification of Cis Acting elements within the Jab1 gene promoter To analyze the mechanisms responsible for transcrip tional regulation of Jab1, luciferase constructs were gen erated to evaluate Jab1 promoter activity.

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