Fig. 4.

Inhibition of NHEJ by NF90 antibody. (A) NF90 is present in complexes with Ku70 and DNA-PKcs in HeLa cells. Immunoprecipitates from HeLa WCE with antibody against Omni (control), NF90, Ku70, NF45, and DNA-PKcs (DPKcs) were assayed by immunoblotting. (B) DNA independence of NF90–DNA-PK interactions. WCE was preincubated with Benzonase (100 U per 300 μg WCE protein) or ethidium bromide (EtBr) before immunoprecipitation with antibody against Omni or NF90 and immunoblotting. (C) Kinetics of in vitro NHEJ reaction using SalI-linearized pUC19 DNA substrate. Oligomeric end-joined species were resolved by agarose gel electrophoresis and quantified with the Image Quant software program. HM, higher multimers (pentamers and above). (D) Inhibition of NHEJ by drugs. Assays contained wortmannin (1, 5, 10, or 20 μM), KU55933 (0.025, 0.05, 0.5, or 1 μM), NU7026 (0.5, 1, 2.5, 5, or 10 μM), or solvent (dimethylsulfoxide [DMSO]; 0.25, 0.5, or 1%). EDTA, supplemented with 50 mM EDTA; −, no additions; M, DNA molecular markers. (E) WCE was treated with Omni, Ku70, DNA-PKcs (DPKcs), or NF90 antibody, left untreated (No Ab), heat inactivated at 90°C, or supplemented with 50 mM EDTA and then assayed for NHEJ activity. (F) Quantitation of residual monomeric substrate (Monomer) and higher multimeric products (higher multimer; pentamers and above) expressed relative to Omni control. Results are means of data from 3 independent experiments in duplicate with standard deviations.