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. 2005 Jul 29;102(32):11296–11301. doi: 10.1073/pnas.0505053102

Fig. 5.

Fig. 5.

Cycling Mad2–/– p53–/– MEFs do not accumulate high levels of double-strand breaks. (A) Indirect immunofluorescence of phospho-H2AX. Mad2+/+ p53–/– (AB103) and Mad2–/– p53–/– (AB98) cells were irradiated with 1,700 rad in a γ cell irradiator. Cells were fixed and stained 30 min after irradiation. –γ, untreated cells; +γ, irradiated cells; DAPI, blue; phospho-H2AX, green. (Scale bar: 10 μm.) Percentage of phospho-H2AX-positive cells is indicated in the images. (B) Quantitation of phospho-H2AX levels in lysates from NIH 3T3 cells, and AB103, AB100, and AB98 MEF lines before and after irradiation. Lysates were prepared from untreated cells and from cells 1 h after irradiation. Immunoblots were probed by using phosphospecific H2AX antibody and monoclonal γ-tubulin antibody as loading control. Phospho-H2AX protein levels were quantitated and normalized to γ-tubulin protein levels by using a FluoroImager459 (Molecular Dynamics) and imagequant software (Molecular Dynamics).