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. 2012 Jun 18;287(33):28122–28131. doi: 10.1074/jbc.M112.368381

FIGURE 4.

FIGURE 4.

Cells expressing FLAG-KAP-1 S473A display defective foci resolution in heterochromatin following DNA damage. A–D, HeLa cells were transfected with FLAG-CMV, FLAG-KAP-1 WT, or FLAG-KAP-1 S473A and treated with 2 Gy of IR before fixation and staining with γH2AX (A and B) or 53BP1 (C and D) antibodies at the indicated times. Representative images are shown in B and D, and graphical representation of data is shown in A and C. The results shown represent the average and S.E. of three independent experiments. The foci in 50–100 cells were counted in each independent experimental condition. * indicates a two-tailed p value of < 0.01. E and F, γH2AX foci that persist in S473A-expressing cells are associated with heterochromatin. Growth-arrested NIH3T3 cells were transfected with FLAG-KAP-1 WT or S473A. After 24 h, cells were mock-treated or treated with 2 Gy of IR and harvested at the indicated times after IR. Cells were stained with γH2AX (green) and DAPI (blue). Foci were scored as heterochromatin-associated positive or negative. The results shown represent the average and S.E. of three independent experiments. The association between γH2AX foci and heterochromatin in 40–50 cells was quantified in each independent experimental condition. ** indicates a two-tailed p value of < 0.0001.