FIG. 3.

(A) Expression analysis of DNA-PKcs PI 3-kinase domain mutants in CHO V3 cells by Western blotting. AA8, wild type; V3, DNA-PKcs mutant; V3-F18, intact DNA-PKcs-transfected V3 cell line; V3-JM, transfection control V3 cell line; V3-KA4, kinase domain II mutant; V3-KB20, domain II mutant; V3-KC23, frameshift mutant which makes the truncated protein; V3-KD51, domain VIb mutant. As a loading control, c-Abl expression was also examined. (B) DNA-activated protein kinase activity of wild-type and mutant DNA-PKcs-expressing cells. DNA-PKcs was immunoprecipitated from whole-cell extracts. Protein kinase activity was analyzed in the absence or presence of added RPA, as indicated. The position of the 32-kDa subunit of recombinant human RPA is indicated. Phosphorylated RPA signals are observed in lane 1 (AA8) and lane 5 (V3-F18). (C) Radiation sensitivity of wild-type and mutant DNA-PKcs-expressing V3 cell lines. V3 cells expressing intact human DNA-PKcs (V3-F18) and each of the V3 cell lines expressing the four kinase domain mutant DNA-PKcs proteins were assayed for radiation sensitivity. Radiation sensitivity of the control pSV2neo transfectant V3-JM is also shown.