53BP1 protein abundance decreases upon irradiation.

<p><b>A</b>. HeLa cells were subjected to 10 Gy X-rays and total cell lysates were prepared in 2% SDS containing buffer at the indicated time points. Immunoblots of indicated protein were shown. H4 was a loading control. The positions of the molecular mass markers in kDa (K) are indicated at the <i>left</i>. <b>B</b>. HA-53BP1 plasmid was transfected into 293T cells, and 48 h post-transfection, cells were irradiated and extracted in SDS containing buffer; NT indicates no transfection (lanes 1–4). Retinal pigment epithelia cells (RPE; lanes 5, 6) and normal mammary epithelial cells (MCF10A; lanes 7, 8) were exposed to 10 Gy X-rays and total cell lysates were prepared in SDS containing buffer 4 h after irradiation. HeLa cells were exposed to 10 Gy irradiation similarly and total cell lysates were extracted 4 h post-IR in buffer containing 2% SDS and 8 M urea (lanes 9, 10). Immunoblots were developed using anti-HA antibody to detect HA-tagged 53BP1 protein in 293T cells (lane 1–4), and antibody specific for 53BP1 to recognize endogenous 53BP1 in RPE, MCF10A and HeLa cells (lane 5–10). <b>C</b>. HeLa cells were subjected to immunofluorescence microscopy 4 h post-irradiation (10 Gy). Cells were stained for 53BP1 (green; <i>top</i>) and merged with DAPI stain of DNA (blue; <i>bottom</i>). <b>D</b>. Similar to the immunofluorescence microscopy experiment in panel C, RPE and MCF10A cells were stained for 53BP1 protein before and after irradiation. For the immunofluorescence images, 100 nuclei were analyzed in HeLa cells, and 30 nuclei were analyzed in RPE or MCF10A cells using ImageJ software. Mean intensity of 53BP1 signal at foci regions in each nucleus in irradiation sample were scored and normalized according to the mean intensity of diffuse 53BP1 signal in no irradiation sample. Results (mean +/− SEM) were shown for three cell lines. <b>E</b>. The distribution of pixel intensities of 53BP1 nuclear stain in HeLa cells, as in panel C, was plotted for No IR (blue) and IR (red). Results were shown from 80–100 nuclei in each sample. Pixel intensities within the range of 850–1400 were shown in the graph inset.