FIG 3.

Cells expressing H2AX K5R are defective in proper NBS1 localization to DNA damage sites. (A) Expression of reconstituted H2AX WT or mutants in MEFs in which the endogenous H2AX was knocked out. (B) Left, examples of microirradiated cells incubated for 4 h before fixation. Microirradiation experiment followed by immunohistochemistry analysis. Top, anti-NBS1 and anti-γ-H2AX antibodies; bottom, anti-MDC1 and anti-γ-H2AX antibodies. Right, fluorescence intensities from the undamaged regions or the damaged regions were quantified and plotted. The average intensities of NBS1 immunofluorescence were derived from 13 cells for H2AX WT-reconstituted cells and 9 cells for H2AX K5R-reconstituted cells, and the average intensities of MDC1 immunofluorescence were derived from 8 cells for H2AX WT-reconstituted cells and 8 cells for H2AX K5R-reconstituted cells. (C) Immunohistochemistry analyses of MEFs reconstituted with H2AX WT, K5R, or S139A following IR (8 Gy, 16 h post-IR). Left, anti-NBS1 and anti-γ-H2AX antibodies; right, anti-MDC1 and anti-γ-H2AX antibodies. (D) The cells with more than 10 foci of NBS1, MDC1, or γ-H2AX were counted among cells irradiated with 8 Gy of IR. Cells were subjected to immunofluorescence analyses either 2 h or 8 h after irradiation. Bar, 10 μm. Graphs show standard deviations.