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Fig. 2. GFP-m53BP1 complements a defect of cells lacking 53BP1. (A,B) Homozygous mutant mice that lack 10 exons of the 53BP1 gene, encoding the last 702 amino acids, were produced (see supplementary material Fig. S1). Western blot analysis of liver, testis and brain extracts from wild-type (+/+) and the mutant (–/–) mice with affinity-purified anti-C-terminus 53BP1 (residues 1255-1957) antibodies (A) or with anti-N-terminus (residues 1-355) antibodies (B). Each well was loaded with 25 µg of the total extracts indicated. The extracts from the mutant mice lack proteins recognized by the antibodies. In A, a faint smear in the –/– brain lane (marked by x) is non-specific binding to a highly expressed protein in the brain that corresponds to the protein band (marked by x) in B, lower panel. (C) Similar analysis was performed in MEF cells. Truncated 53BP1 (Tr) is detected in the total extracts of MEF cells from the mutant mice, when anti-N-terminus serum is used. The amount of the truncated protein is significantly lower than the wild-type protein (WT). Preimmune serum for the anti-N-terminus did not react with any proteins. (D) MEF cells were exposed to 10 Gy of X rays, allowed to recover for 1 hour, fixed and subjected to staining with the antibodies indicated. In contrast to the wild-type 53BP1, which shows focal distribution, the truncated 53BP1 in mutant MEFs (–/–) does not show focal distribution when probed with anti-N antibodies. DNA was counterstained with DAPI. (E) GFP-m53BP1 fusion protein (green) was transiently expressed in the mutant MEF (–/–), irradiated with 10 Gy X rays, fixed and stained with anti-phospho-Chk2 antibodies (Chk2T68P; Cy3 in red). Bar, 10 µm.
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