FIG. 6.

Defects in V(D)J-initiated DNA repair, chromosome instability, and impaired DDR in Nbs1-deleted T cells. (A) ChIP analysis using antibodies against Rad50, γ-H2AX, and Ac-H4. These antibodies recovered distal and proximal Vβ (left) and Vα (right) regions of Nbs1^T-ctr and Nbs1^T-del thymocytes. Co, Nbs1^T-ctr; Δ, Nbs1^T-del; γ-H2AX, phospho-histone H2AX; Ac-H4, acetyl-histone H4. (B) Summary of the chromosomal stability in Nbs1^T-ctr and Nbs1^T-del T cells. The metaphases were prepared from 8-week-old Nbs1^T-ctr and Nbs1^T-del T cells 48 h after IL-2/PMA/ionomycin stimulation. n, number of metaphases analyzed. **, P < 0.01. Student's unpaired t test was used, except that the chi-square test was applied for calculation of the percentage of metaphases containing aberrations. (C) Western blot analysis of Nbs1 and Chk2 in whole-cell extracts from Nbs1^T-ctr and Nbs1^T-del thymocytes after 2 h of treatment with 0.2 μg/ml of adriamycin (Adr). PARP-1 is used as a loading control. The ratio of the phosphorylated form of Chk2 (upper band) was corrected to that of the nonphosphorylated form (lower band) by ImageJ software and indicated under the corresponding lanes. NA, not applicable.