Figure 5.
Figure 5. Constitutive increase in Ku70/80 heterodimer DNA-end binding activity. (A) Ku DNA-end binding activity in whole protein extracts from resistant (R) and sensitive B-CLL (S) cells (1 μg/lane) at the indicated times after treatment with 10 Gy γ-irradiation. The results shown are representative of those obtained in all extracts tested (n = 3 for resistant samples and n = 3 for sensitive samples). (B) Ku DNA-end binding activity (mean ± SEM in arbitrary units, AU) in 3 resistant B-CLL samples (R) and 3 sensitive B-CLL samples (S) at the indicated times after irradiation. (C) Untreated whole cell protein extracts (1 μg/lane) from the Ku80-deficient xrs6 cell line and the CHO-K1 line (expressing both Ku70 and Ku80 proteins). MO59J (DNA-PKcs deficient) and MO59K (normal levels of DNA-PKcs activity) were used as controls. (D) Untreated B-CLL lymphocyte protein extracts (5 μg, lane 1) were immunodepleted using 125 ng (lane 2) and 250 ng (lane 3) monoclonal Ku70 antibody, assayed for Ku DNA-end binding activity (1 μg protein extract/lane) (upper panel) and then subjected to Western blot (WB) analysis (lower panel). For supershift assays (lanes 4-5), samples were treated with 5 μg untreated B-CLL lymphocyte protein extract (lane 5) or with 250 ng polyclonal Ku70 antibody (lane 4), and then assayed for Ku DNA-end binding (1 μg of protein extract/lane). (E) Whole-cell extracts (5 μg) were obtained from representative sensitive and resistant B-CLL cell samples at the indicated times after irradiation. Extracts were subjected to electrophoresis on a 10% SDS polyacrylamide gel (DNA-PKcs analysis was carried out using a 6% SDS polyacrylamide gel). Proteins were then blotted onto polyvinylidenefluoride (PVDF) membranes and were detected using the appropriate antibodies. (F) Whole-cell extracts (5 μg) from 6 sensitive (S) and 4 resistant (R) untreated B-CLL cell samples, and 4 untreated normal B-cell samples from healthy donors (B cells) were subjected to Western blotting analysis as described in panel E. See Supplemental Figure S3 for more information.

Constitutive increase in Ku70/80 heterodimer DNA-end binding activity. (A) Ku DNA-end binding activity in whole protein extracts from resistant (R) and sensitive B-CLL (S) cells (1 μg/lane) at the indicated times after treatment with 10 Gy γ-irradiation. The results shown are representative of those obtained in all extracts tested (n = 3 for resistant samples and n = 3 for sensitive samples). (B) Ku DNA-end binding activity (mean ± SEM in arbitrary units, AU) in 3 resistant B-CLL samples (R) and 3 sensitive B-CLL samples (S) at the indicated times after irradiation. (C) Untreated whole cell protein extracts (1 μg/lane) from the Ku80-deficient xrs6 cell line and the CHO-K1 line (expressing both Ku70 and Ku80 proteins). MO59J (DNA-PKcs deficient) and MO59K (normal levels of DNA-PKcs activity) were used as controls. (D) Untreated B-CLL lymphocyte protein extracts (5 μg, lane 1) were immunodepleted using 125 ng (lane 2) and 250 ng (lane 3) monoclonal Ku70 antibody, assayed for Ku DNA-end binding activity (1 μg protein extract/lane) (upper panel) and then subjected to Western blot (WB) analysis (lower panel). For supershift assays (lanes 4-5), samples were treated with 5 μg untreated B-CLL lymphocyte protein extract (lane 5) or with 250 ng polyclonal Ku70 antibody (lane 4), and then assayed for Ku DNA-end binding (1 μg of protein extract/lane). (E) Whole-cell extracts (5 μg) were obtained from representative sensitive and resistant B-CLL cell samples at the indicated times after irradiation. Extracts were subjected to electrophoresis on a 10% SDS polyacrylamide gel (DNA-PKcs analysis was carried out using a 6% SDS polyacrylamide gel). Proteins were then blotted onto polyvinylidenefluoride (PVDF) membranes and were detected using the appropriate antibodies. (F) Whole-cell extracts (5 μg) from 6 sensitive (S) and 4 resistant (R) untreated B-CLL cell samples, and 4 untreated normal B-cell samples from healthy donors (B cells) were subjected to Western blotting analysis as described in panel E. See Supplemental Figure S3 for more information.

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