Fig. 2

RIF1 interacts with CSB and recruits CSB to DSBs via its CTD domain. a Schematic diagram of RIF1. NLS: nuclear localization signal; CTD: C-terminal domain. b Schematic diagram of CSB. c Quantification of the percentage of cells exhibiting Myc-CSB accumulated at the lac operator array. U2OS-265 cells were co-transfected with Myc-CSB and various mCherry-LacR-RIF1 alleles as indicated. A total of 250 cells positive for Myc-CSB expression were scored for each independent experiment in a blind manner. SDs from three independent experiments are indicated. d Quantification of the percentage of cells exhibiting Myc staining accumulated at the lac operator array. U2OS-265 cells were co-transfected with mCherry-LacR-RIF1-C and various Myc-tagged CSB alleles as indicated. A total of 250 cells positive for expression of various Myc-tagged CSB alleles as indicated were scored for each independent experiment in a blind manner. SDs from three independent experiments are indicated. e Quantification of the percentage of cells exhibiting Myc-CSB-C accumulated at the lac operator array. U2OS-265 cells were co-transfected with Myc-CSB-C and various mCherry-LacR-RIF1-C alleles as indicated. A total of 250 cells positive for Myc-CSB-C expression were scored for each independent experiment in a blind manner. SDs from three independent experiments are indicated. f Coimmunoprecipitation with anti-Myc antibody in 293T cells expressing the vector alone or Myc-CSB-C in conjunction with various mCherry-LacR-CTD alleles as indicated. Immunoblotting was done with anti-Myc and anti-mCherry antibodies. g Quantification of the intensity of Myc-CSB signal at the site of FokI-induced DSBs. 24 h post transfection with siControl or siRIF1, U2OS-265 cells were transfected with the EGFP vector alone or various siRIF1-resistant EGFP-RIF1 alleles as indicated and induced for FokI expression 48 h post transfection. Analysis of Myc-CSB signal intensity was only done for cells positive for expression of Myc-CSB, EGFP and mCherry-LacR-FokI. The respective numbers of cells analyzed for siControl/EGFP, siRIF1/EGFP, siRIF1/EGFP-RIF1 and siRIF1/EGFP-Rif-DCTDIII were 131, 107, 120 and 124. *P < 0.05 (Mann–Whitney test). NLS nuclear localization signal; UBD ubiquitin-binding domain; WHD winged helix domain