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Fig. 1. Dynamics of GFP-m53BP1 in vivo. (A) HeLa cells stably expressing GFP fused to full-length mouse 53BP1 (GFP-m53BP1) were untreated or exposed to ionising radiation (IR; 10 Gy of X rays). After recovery for 1 hour, cells were fixed and stained for phosphorylated histone H2AX (
H2AX). (B) Extracts were prepared from the HeLa cell line stably expressing GFP-m53BP1 and analysed by western blotting with anti-GFP antibodies (lanes 1, 2) and with antibodies against the C-terminus of mouse 53BP1 (lanes 3,4,5). Equal amounts of extracts from mouse NIH3T3 cells (lane 3) and un-transfected HeLa cells (lanes 1, 4) were run as controls. Samples shown here were separated on the same gel. GFP-m53BP1 migrates slightly slower than the endogenous m53BP1, which makes the band in lane 5 broader than the band in lane 4. Note that the two proteins were not separable. (C) Accumulation of GFP-m53BP1 at the nuclear region damaged by high power laser. Wild-type MEF (mouse embryonic fibroblast) cells were transfected with GFP-m53BP1 and irradiated with a laser at the region indicated in red. After GFP-m53BP1 stripe formation, the cells were fixed and stained for phosphorylated () H2AX with Cy3 (red). The bright spots stained with DAPI are condensed heterochromatin of mouse cells. (D) Time lapse recording of laser-induced stripe formation (the stripe is marked by an arrowhead). Wild-type MEF cells, transfected with GFP-m53BP1 or GFP-M, were micro-irradiated and maintained at 37°C. Sequential images were captured and used to create a movie series. Selected frames are shown, along with the corresponding time (in seconds) after micro-irradiation.
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