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. 2012 Jul 25;287(38):31747–31756. doi: 10.1074/jbc.M112.384750

FIGURE 3.

FIGURE 3.

The Rad50 zinc hook domain is important for H2AX-independent binding of MRN to chromosomal DSBs. A, interaction of MRN complex with MDC1. Myc-tagged Mre11 and Nbs1 with either Myc-tagged Rad50WT or Rad50 hook mutants Rad50HK-AA and Rad50HK-NA was co-transfected with HA-MDC1 or control (−) in 293T cells. Anti-HA immunoprecipitation (IP) was carried out in whole cell lysates and followed by Western blotting with indicated antibodies. B, recruitment of Rad50 in H2AX knockdown cells. EGFP-Rad50WT or EGFP-Rad50HK-NA was transfected into U2OS cells, with endogenous H2AX silenced by EBFP-marked H2AX shRNAs. The absolute intensities of Rad50 recruitment to the damage sites after laser microirradiation were determined. Western blot shows silencing of endogenous H2AX, with Ku70 used as a loading control. C, recruitment of Nbs1 in H2AX-deficient cells. mRFP-Nbs1 was transfected in U2OS cells with either mock (Ctrl) or H2AX knockdown (H2AXsh) cells. DNA damage was introduced by laser microirradiation. Absolute intensities of Nbs1 recruitment were determined at the indicated time points.