Ku80, Ku70 and NF90 bind specifically and dynamically to the IL-2 promoter

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in activated T-cells"</p><p></p><p>Nucleic Acids Research 2007;35(7):2302-2310.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874627.</p><p>© 2007 The Author(s)</p> Jurkat T-cells or mouse primary spleen cells were NS or stimulated for 4 hr with PMA + ionomycin (S) then nuclear proteins were crosslinked to chromatin with 1% formaldehyde. () Sheared and restricted chromatin was used as template for PCR amplifications (35 cycles) of IL-2 intron 3 (negative control), IL-2 proximal promoter, myc1 (negative control) and myc11 (origin of DNA replication, positive control for Ku binding) sequences. () Ku80 chromatin immunoprecipitation (ChIP) was performed using mAB 111. () Ku70 ChIP was performed using polyclonal antibody. () NF90 ChIP was performed using mAB DRBP76. () Input chromatin from mouse spleen cells was used as template for amplification of IL-2 proximal promoter, IL-2 intron 3 (negative control) and adenosine deaminase origin of DNA replication (positive control for Ku binding). () Ku80 ChIP. () Ku70 ChIP. () NF90 ChIP. () Jurkat T-cells were NS or stimulated for 4 hr with PMA + ionomycin (P/I) or stimulated in the presence of immunosuppressants cyclosporin A (P/I/CsA) or triptolide (P/I/Trip), then IL-2 promoter ChIP was performed, using specific antibodies against Ku80, Ku70 and NF90 and 30 cycles of PCR amplification