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. 2016 Nov 14;291(53):26987–27006. doi: 10.1074/jbc.M116.751867

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© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Author's Choice—Final version free via Creative Commons CC-BY license.

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FIGURE 3. — Integrity of NHEJ complexes on SEC. A and B, SEC profiles of APLF, Ku, DNA-PKcs, Ku·APLF, Ku·DNA-PKcs, Ku·DNA-PKcs·APLF, and Ku·X4L4 in the presence of 20bpDNA are colored as indicated. SEC profiles are normalized at the maxima of the main peak. B, zoom in of the SEC profiles of the complexes shown in panel A. C, SDS-PAGE of APLF SEC-peak fractions together with stock solution of APLF (lane 1) and Ku heterodimer (lane 2). Note that APLF and Ku chain Ku70 run at the same level. D, SDS-PAGE of Ku/APLF SEC-peak fractions in the presence of 20bpDNA shows the presence of Ku70, Ku80, and APLF as indicated. E, Ku·20bpDNA·APLF SEC fractions 1 and 2 (lanes 6 and 7) from panel D together with positive and negative controls as indicated were boiled in SDS sample buffer, loaded onto SDS-PAGE gels, and immunoblotted with antibodies to APLF. F–H, SDS-PAGE of DNA-PKcs·Ku, DNA-PKcs·Ku·APLF, and Ku·X4L4·APLF SEC, peak fractions in the presence of 20bpDNA shows the presence of the complex components as indicated. I, the most concentrated fraction, 1, of Ku·20bpDNA·X4L4·APLF from panel H (lane 2) together with positive control were boiled in SDS sample buffer, loaded onto SDS-PAGE gels, and immunoblotted with antibodies to APLF.