Each Cells were lysed by the addition of high salt lysis buffer directly to the plates as described in the previous section, and the lysates were processed as previously described

The major antibodies used were: goat polyclonal anti-topo I and goat polyclonal anti-ARF C-terminus (Santa Cruz Biotechnology, Santa Cruz, CA) mouse monoclonal anti-phosphoserine, mouse monoclonal anti-FLAG, and rabbit polyclonal antiFLAG (Sigma-Aldrich, St. Louis, MO) rabbit polyclonal antihistone H2A.X and rabbit polyclonal anti-topo I (Bethyl Laboratories, Montgomery, TX) rabbit polyclonal antiH2A.X [ser139] (Novus Biologicals, Littleton, CO) mouse monoclonal anti-histone H3 (Millipore, Temecula, CA) and rabbit polyclonal anti-PS506 (pAb506-P described in reference [23]). The secondary antibodies employed were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, goat anti-mouse IgGHRP, and donkey anti-goat IgG-HRP (all from Santa Cruz Biotechnology). For Westerns, main and secondary antibodies were utilised at 1:a hundred and one:1000 dilutions, respectively.The entire-length human topo I sequence was PCR amplified from H358 cDNA and subcloned into the pCMV-Tag1 vector (Pefabloc FG Stratagene, San Diego, CA), which materials an N-terminal FLAG sequence. The cloned insert was sequenced (Retrogen, San Diego, CA) and confirmed to be the human wild-sort topoisomerase I coding sequence (NM_003286). The cloned full-length sequence was then used as a template to PCR amplify a sequence of C-terminal deletion mutants of various lengths, the main region sequence (residues 21636), and the location from residues 5012765 that contains element of the main domain, the linker, and the topo I Cterminus. The amplified deletion fragments had been also inserted into the pCMV-Tag1 Vector (Stratagene) and all cloned inserts ended up then subcloned collectively with the FLAG sequence into the pTriExTM-two Hygro vector (Novagen), which supplies a His-tag and T7 promoter for in vitro transcription/translation. Each Cells have been lysed by the addition 22505653of large salt lysis buffer straight to the plates as explained in the preceding part, and the lysates ended up processed as formerly explained [fourteen].