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. 2012 Jan;32(1):76–87. doi: 10.1128/MCB.05661-11

Fig 3.

Fig 3

Ku70 S155A/D156A mutation does not affect DNA repair. (A) The S155A/D156A mutation does not interfere with the repair of IR-induced genomic DNA damage. PFGE analysis was performed on genomic DNA from Ku70−/− MEFs expressing wild-type Ku70 (WT), Ku70 S155A/D156A, or empty pMSCV either untreated (control) or immediately after IR treatment (0 h) or 2 or 6 h following IR treatment. For all samples, FAR (fraction of activity released) was averaged from three independent experiments, with error bars representing standard errors of the means. (B) The Ku S155A/D156A mutation does not interfere with the repair of extrachromosomal DNA breaks. Ku70−/− MEFs expressing wild-type Ku70 (WT), mutant S155A/D156A, or empty vector (pMSCV) were transfected with a linearized pGL3-Promoter plasmid and control pRL-SV40 plasmid and assayed for luciferase activity 48 h later. Data represent the average firefly luciferase values normalized to the renilla luciferase values for three separate experiments, with error bars representing SD (*, P < 0.01). (C) Ku70 S155A/D156A mutation rescues the IR sensitivity conferred by the D192A/D195R substitution. Clonogenic survival assay of Ku70−/− MEFs expressing wild-type Ku70 (WT), Ku70 bearing the substitutions D192A/D195R, or the double mutant D192A/D195R, S155A/D156A. Results are averaged from three experiments, and the error bars represent the SD. (D) Western blot analysis of Ku70−/− MEFs with empty vector (pMSCV) or Ku70−/− MEFs expressing wild-type Ku70 (WT) and Ku70 mutants as indicated. The blot was analyzed with antibodies to Ku80, Ku70, and actin. (E) Ku70 S155A/D156A substitutions do not rescue the DNA repair defect conferred by the Ku70 D192A/D195R mutation. PFGE analysis was done as described for panel A with genomic DNA from Ku70−/− MEFs expressing wild-type Ku70 (WT), D192A/D195R, the double mutant D192A/D195R, S155A/D156A, or empty pMSCV either untreated (control) or immediately after (0 h) and 6 h following IR treatment. FAR was averaged from three independent experiments, with error bars representing SEM (∗, P < 0.05).