FIGURE 1.

R1 is required for DNA·PK·PP6c interaction. A, schematic representation of the three possible modes of DNA-PK-R1-PP6c interaction. i, DNA-PK binds to PP6c directly, and R1 in the complex is bound to only to PP6c and not to DNA-PK. ii, R1 binds to both DNA-PK and PP6c, thus bringing DNA-PK and PP6c in close proximity. iii, DNA-PK binds to both PP6c and R1 simultaneously. B, 293T cells were transfected with control (non-target) or R1 siRNA (R1). Seventy-two hours post-transfection, endogenous PP6c was immunoprecipitated (IP) from the lysates of these cells using anti-PP6c antibody. The levels of input proteins (top four panels) and immunoprecipitated proteins (bottom two panels) were determined by Western blotting with the indicated antibodies. 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 non-targeting siRNA (first lane). Values below the Western blot analysis are mean ± S.D. from three independent experiments. p < 0.001. C, 293T cells were transfected with empty vector (vector) or the indicated HA-tagged PP6c constructs. Forty-eight hours post-transfection, HA-tagged PP6c was immunoprecipitated from the lysates of these cells using anti-HA antibody. The levels of input proteins (top three panels) and immunoprecipitated proteins (bottom three panels) were determined by Western blotting with the indicated antibodies. 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 HA-PP6c-WT (second lane). Values below the Western blot analysis are mean ± S.D. from three independent experiments. p < 0.001 for both HA-PP6c-H114A and HA-PP6c-HR-QA.