Native DNA-PKcs and Ku70/80 were purified to homogeneity from HeLa cells as described previously (19 (link)). The EMSA binding reactions were carried out in 10 μl volume containing 100 mM NaCl, 10 mM Tris, pH 7.5, 5% (w/v) glycerol, 1 mM MgCl2, 0.8 U of RNAse OUT (Invitrogen) and 0.33 pmol of 32P-labeled hTR probe. The amount of purified proteins used in each EMSA reaction is indicated in the figure legends. Following a 10 min incubation at 25°C, the reactions were fractionated on 5% non-denaturing PAGE, in 0.5× TBE for 90 V for 3 h. For the smaller truncated hTRs, the gels were run for 1 h.
For the EMSA experiments with DNA, a double-stranded 36 bp blunt-ended oligonucleotide corresponding to the sequence of a non relevant gene (PIG3) was used as the radiolabeled probe. The DNA probe was end-labeled with 32P using T4 kinase as described previously (20 (link)), and approximately 0.2 pmol of labeled probe was used for the binding reactions. For the DNA gel shift, the gels were run for 1 h.
The EMSA gels were dried on Whatman 3MM paper and exposed to X-ray films. Addition of competitor RNA, DNA or antibodies to the EMSA reactions, either before or 5 min after the radiolabeled probe, did not alter the results. The monoclonal antibodies used included anti-Ku70 and anti-Ku80 (Ab5 and Ab2, NeoMarkers) and anti-Myc (Santa Cruz Biotechnology).
For the EMSA experiments with DNA, a double-stranded 36 bp blunt-ended oligonucleotide corresponding to the sequence of a non relevant gene (PIG3) was used as the radiolabeled probe. The DNA probe was end-labeled with 32P using T4 kinase as described previously (20 (link)), and approximately 0.2 pmol of labeled probe was used for the binding reactions. For the DNA gel shift, the gels were run for 1 h.
The EMSA gels were dried on Whatman 3MM paper and exposed to X-ray films. Addition of competitor RNA, DNA or antibodies to the EMSA reactions, either before or 5 min after the radiolabeled probe, did not alter the results. The monoclonal antibodies used included anti-Ku70 and anti-Ku80 (Ab5 and Ab2, NeoMarkers) and anti-Myc (Santa Cruz Biotechnology).
