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. 2012 Feb 1;287(12):9230–9239. doi: 10.1074/jbc.M111.333708

FIGURE 5.

FIGURE 5.

Residues 1–326 of R1 are sufficient for binding DNA-PK. A, schematic representation of the FLAG-tagged constructs of R1 used. F, N-terminal FLAG tag; H, C-terminal 6X-histidine tag. B, 293T cells were transfected with empty plasmid or plasmids encoding the FLAG-tagged proteins described in A. Forty-eight hours post-transfection, FLAG-tagged proteins were immunoprecipitated (IP) from the lysates of these cells using anti-FLAG antibody. The levels of input proteins (top four panels) and immunoprecipitated proteins (bottom three panels) were determined by Western blotting with the indicated antibodies. The arrows indicate the position of FLAG-tagged proteins. The relative amount of coprecipitated DNA-PK was quantified by densitometric analysis and compared with the amount of DNA-PK coprecipitated from cells transfected with FLAG-R1 (second lane). Values below the Western blot are mean ± S.D. from three independent experiments. For FLAG-1–521, FLAG-1–521-HIS, and FLAG-1–326-HIS, p < 0.05. C, schematic representation of the FLAG-tagged constructs of R1 used. The asterisks indicate the position of the charge reversal mutations. The charge reversal mutants are denoted as 4DE-KK. D, 293T cells were transfected with empty plasmid or plasmids encoding the FLAG-tagged proteins described in C. Forty-eight hours post-transfection, FLAG-tagged proteins were immunoprecipitated from the lysates of these cells using anti-FLAG antibody. The levels of input proteins (top two panels) and immunoprecipitated proteins (bottom two panels) were determined by Western blotting with the indicated antibodies. The arrows indicate the position of FLAG-tagged proteins. The relative amount of coprecipitated DNA-PK was quantified by densitometric analysis and compared with the amount of DNA-PK coprecipitated from cells transfected with FLAG-1–521-HIS (lane 2). Values below the Western blot are mean ± S.D. from three independent experiments. For FLAG-1–521-HIS (4DE-KK) and FLAG-1–326-HIS (4DE-KK), p < 0.005; difference not significant for FLAG-1–326-HIS.