Cisplatin-induced recruitment of FACT to the Ku complex. A. Purification of the Ku86 complex. Tandem affinity purification for FLAG and HA was performed on nuclear extracts from S3 or S3-Ku86-Flag/HA cells 0, 1, 2 and 4 hours (hrs) after cisplatin (CIS) treatment. The purified complexes were resolved by 4-12% NuPAGE and visualized by silver staining. Arrows show Ku70, Ku86-Flag/HA, the band identified as spt16 through MSMS analysis and DNA-PKcs (based on molecular weight). The molecular weight markers are indicated on the left (in kDa). B. Association of FACT with the Ku complex. Complexes purified as in (A) before or 4 hrs after cisplatin treatment (+CIS) were immunoblotted for the indicated proteins. Cells were pre-treated with the DNA-PK inhibitor Nu7026 as indicated. C. Cisplatin-induced association of γH2AX with the Ku complex. Inputs are nuclear extracts (NEX) and chromatin obtained before or 4 hrs after cisplatin treatment (+CIS). Inputs and Ku86 complexes purified from the inputs were immunoblotted for the indicated proteins. D. Treatment of the Ku complex with DNase reveals DNA-dependent interactions. Ku86 complexes were purified as in (A) 2 hrs after cisplatin treatment. The complexes immobilized on anti-HA beads were left untreated or treated with DNase and then eluted with the HA peptide. The resulting complexes were analyzed by immunoblotting for the indicated proteins.