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. 2013 May 27;201(5):693–707. doi: 10.1083/jcb.201302145

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© 2013 Reczek et al.

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Figure 6. — Ctip^S326A/S326A MEFs are proficient for assembly of Rad51 and RPA nuclear foci in response to DNA damage. (A and B) Ctip^+/+ and Ctip^S326A/S326A MEFs were exposed to IR (10 Gy) and IRIF formation was assessed 1 h later by immunostaining with rabbit antisera specific for Rad51 (A) or Thr21-phosphorylated RPA2 (B). Cells containing 10 or more distinct Rad51- or phosphorylated RPA2-staining nuclear foci were counted in at least 500 nuclei of two independent MEF lines for each genotype, and the error bars represent SEM. IR treatment strongly induced the number of Rad51 foci in Ctip^+/+ and Ctip^S326A/S326A MEFs, but not in Brca1^{S1598F/S1598F} MEFs, which are known to have reduced IRIF assembly of Rad51 (Shakya et al., 2011). IR treatment also strongly induced the number of Thr21-phosphorylated RPA2 IRIFs in Ctip^+/+ and Ctip^S326A/S326A MEFs, as well as in Brca1^{S1598F/S1598F} MEFs. (C) Independent clones of Ctip^+/+ and Ctip^S326A/S326A MEFs were cultured in the presence or absence of 1.0 µM camptothecin (CPT) and harvested 1 h later. Total cell extracts of each culture were then fractionated by SDS-PAGE and immunoblotted with antibodies specific for Ctip, Ser4/Ser8-phosphorylated RPA2, total RPA2, or α-tubulin.