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. 2015 Apr 27;43(10):4962–4974. doi: 10.1093/nar/gkv369

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© The Author(s) 2015. 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/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — The single-strand binding protein complex, hSSB1/2-INTS3 recruits ATRIP to single-stranded DNA. (A) Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-tagged non-specific control protein (HA-NS) were individually expressed in 293T cells, purified by immunoprecipitation with anti-Myc or anti-HA antibodies and were eluted with Myc or HA peptides. For the single-stranded DNA-binding assay, streptavidin-agarose was incubated with non-biotinylated (lane 2) or biotinylated (lane 3) single-stranded DNA followed by incubation with Myc-hSSB1 and Myc-INTS3. Next, HA-ATRIP or HA-NS purified from 293T cells were incubated with streptavidin-agarose bound biotinylated ssDNA either in the absence (lanes 4 and 6) or presence (lanes 5 and 7) of bound Myc-hSSB1 and Myc-INTS3. After washing, the bound proteins were identified by immunoblotting with anti-HA (top panel) and anti-Myc (bottom panel) antibodies. 10% of NS and ATRIP utilized for binding to streptavidin-agarose has been shown in lanes 1 and 8 respectively and specific proteins have been marked by arrowheads. The control protein (NS) did not bind to hSSB1-INTS3 complex, ruling out non-specific association. The numbers indicate relative binding of HA-ATRIP to ssDNA in the absence or presence of Myc-hSSB1 and Myc-INTS3. *P-value was calculated using two-tailed t-test which displays that the ATRIP binding observed in the absence or presence of hSSB1-INTS3 complex is significantly different (*P-value = 0.043). (B) RPA complex is absent in Myc-hSSB1 and Myc-INTS3 immunoprecipitates. Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-NS proteins expressed in 293T cells and purified by elution with Myc or HA peptides following immunoprecipitation were immunoblotted with anti-RPA70 (top panel) and anti-RPA32 (second panel) antibodies for detecting endogenous RPA70 and RPA32. As reported earlier, RPA complex physically associates with ATRIP but is absent from hSSB1-INTS3 complex. Note the high sensitivity of detection of endogenous RPA70 and RPA32 in 293T cell lysate. HA-ATRIP (hollow arrowhead), HA-NS (black arrow) Myc-INTS3 (black arrowhead) and Myc-hSSB1 (shaded arrowhead) have been marked.