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. 2020 Mar 25;3:144. doi: 10.1038/s42003-020-0870-z

Fig. 6. Expression of NHEJ1 and DSBs in the Cp mutant.

Fig. 6

a Expression of an EYFP-tagged chicken NHEJ1 gene and an EYFP-tagged Cp-derived mutant NHEJ1 gene in chicken primary cells. Extracts from the wild-type chicken primary cells transiently expressing the EYFP-chicken NHEJ1 chimeric protein (EYFP-NHEJ1), EYFP-chicken NHEJ1 mutant chimeric protein (EYFP-NHEJ1mt), or control protein (EYFP) were prepared and subjected to Western blotting using anti-GFP, anti-NHEJ1, or anti-β-actin antibodies. b Imaging of the EYFP-NHEJ1-transfected chicken primary cells before and after 405 nm laser microirradiation. Expression of EYFP-NHEJ1, differential interference contrast images (DIC), and merged images are shown. EYFP-NHEJ1 accumulates immediately at DSBs induced by laser microirradiation. Arrowheads show the microirradiated sites. c Immunostaining of microirradiated EYFP-NHEJ1-expressing cells with an anti-γH2AX antibody. The cells were fixed and stained with the anti-γH2AX antibody 5 min after microirradiation. Expression of EYFP-NHEJ1, immunostaining with the anti-γH2AX antibody, and merged image are shown. d Time-dependent EYFP-NHEJ1 accumulation in a living cell 5–120 s after microirradiation. e Primary cells that express EYFP-NHEJ1mt or EYFP before and after microirradiation. Arrowheads show the microirradiated sites. The EYFP-NHEJ1mt is mislocalized in cells and cannot accumulate at DSB sites. f Summary of the results presented. Nuc, nucleus; Cyt, cytoplasm. g Comparison of γH2AX expression in the wild-type (+/+, WT) and mutant (Cp/Cp) chicken cells from the GSP/Cp line. The cells were X-irradiated with 2 Gy, and immunostaining patterns of γH2AX were examined 1 h and 24 h after X-irradiation. Nuclear DNA was stained with DAPI. The γH2AX expression remains even after 24 h in the mutant (Cp/Cp) chicken cells. All scale bars indicate 10 μm.