Irdye 800cw goat anti mouse immunoglobulin g

RADAR protocol was performed as described (48 ). Cells (4.5 × 10⁵) were seeded in six-well plates and then treated according to the experimental scheme. After CPT treatment, the medium was removed, and cells were lysed with 1 ml of DNAzol (10503027, Thermo Fisher Scientific) and collected by scraping. Genomic DNA and DNA-protein covalent complexes were precipitated at −20°C by addition of 0.5 volume of 100% cold ethanol, recovered by centrifugation at 10000 rpm at 4°C, washed twice in 70% ethanol, and resuspended in 300 μl of freshly prepared 8 mM NaOH after air drying for 3 min. The DNA content was measured with a NanoDrop One instrument (Thermo Fisher Scientific), normalized in 1 ml of tris-buffered saline buffer at 10 ng/μl, and measured again before starting with the slot blotting procedure. DNA was then deposited onto a 0.45-μm nitrocellulose membrane (Amersham) using a slot blotting apparatus (1706542, Bio-Rad). TOP1-ccs were detected by overnight incubation with primary rabbit anti-TOP1 antibody (1:2000; ab28432, Abcam, RRID:AB_778545) and secondary IRDye 800CW goat anti-mouse immunoglobulin G (926-32210, LI-COR). Images were acquired with Odyssey CLx Imager (LI-COR), and TOP1-ccs were quantified by ImageStudio Software (RRID: SCR_013715) and are expressed as fold change normalized on the untreated WT sample.