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. 2016 Sep 30;44(22):10758–10771. doi: 10.1093/nar/gkw876

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 2. — FANCD2 and FANCI interact with RAD51. (A) Pull-down assay with the HeLa cell extracts. The beads without RAD51 (N) or with RAD51 (R) were incubated with the nuclease-treated cell extracts, and the proteins bound to the beads were detected by Western blotting. The cell extracts were prepared from untreated and MMC-treated HeLa cells. (B) Pull-down assay with the DT40 cell extracts. The nuclease-treated cell extracts were prepared from untreated, MMC-treated and HU-treated DT40 cells. (C) Pull-down assay with Ni-NTA beads. RAD51 bound to His₆-tagged FANCI, FANCD2 and the I-D complex was copelleted with the Ni-NTA beads, and the proteins were analyzed by SDS-PAGE. (D) Pull-down assay with single-stranded DNA (ssDNA) beads. The beads conjugated with ssDNA, the RAD51-ssDNA complex or the RPA-ssDNA complex were incubated with the I-D complex. The I-D complex that copelleted with the beads was analyzed by SDS-PAGE. The amounts of the I-D complex in the bound fractions were quantitated, and the mean percentages of three independent experiments are indicated with the standard deviations. (E) Pull-down assay with His₆-tagged RAD51 proteins. The I-D complex was copelleted with His₆-RAD51 or His₆-RAD51 F86E bound to Ni-NTA beads, and the proteins were analyzed by SDS-PAGE with Coomassie Brilliant Blue staining. The amounts of the I-D complex in the bound fractions were quantitated, and the mean percentages of three independent experiments are indicated with the standard deviations.