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. Author manuscript; available in PMC: 2010 Jan 13.
Published in final edited form as: Mol Cell. 2008 Feb 29;29(4):488–498. doi: 10.1016/j.molcel.2007.12.029

Figure 6. Bcl2 directly disrupts the Ku/DNA-PKcs complex.

Figure 6

(A) The Ku 70/DNA-PKcs or Ku 86/DNA-PKcs complex was co-immunoprecipitated from H1299 parental cells and incubated with purified Bcl2 (10–20ng/ml) in 1% CHAPS lysis buffer at 4 °C for 1h. After centrifugation, the resulting supernatant and immuno-complex beads were subjected to SDS-PAGE. Ku, Bcl2 and DNA-PKcs that bound to Ku or unbound DNA-PKcs present in the supernatant were then analyzed by Western blot. (B) The Ku 70/DNA-PKcs or Ku 86/DNA-PKcs complex was incubated with linearized DNA in the absence or presence of purified WT Bcl2 (20ng/ml) in 1% CHAPS lysis buffer at 4 °C for 1h. The Ku/DNA-PKcs complex was analyzed as described above. (C) H1299 cells expressing WT or each of the BH deletion Bcl2 mutants were disrupted in 1% CHAPS lysis buffer. Co-IP was performed using an agarose-conjugated Ku 70 or Ku 86 antibody, respectively. The Ku-associated DNA-PKcs was then analyzed by Western blot. Rabbit preimmune serum (Pre) was used as a control.