Fig. 8 | Cell Death & Differentiation

Fig. 8

From: βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1

Fig. 8

βarr1 forms a complex with 53BP1 and influences the DNA damage response also in human cells. a Co-immunoprecipitation (co-IP) experiments were performed from cell lysates derived from U2OS cells endogenously expressing βarr1 and 53BP1. Clarified lysates were incubated with either anti-βarr1 (K-16) antibody, or anti-53BP1 antibody or IgG and the resulting products resolved by SDS-PAGE and probed by western blot analysis using antibodies directed against 53BP1 and βarr1. b 53BP1 and βarr1 expression in cytosol and nucleus. c Cell lysates derived from WT U2OS, U2OS/Cas9/ARRB1-gRNA1 clone 1 (βarr1−/− #1) and U2OS/Cas9/ARRB1-gRNA1 clone 11 (βarr1−/− #11) were immunoblotted and analyzed using the indicated antibodies. βarr1−/− #11 was selected for further assays. d Representative confocal immunofluorescence microscopy images of WT and βarr1−/− U2OS cells 10 min after 4 Gy IR, probed with anti-53BP1 or anti-γ-H2AX antibodies. Merged image reveals colocalization of 53BP1 foci with γ-H2AX foci and increased 53BP1-containing DNA-repair foci formation in βarr1−/− U2OS compared with WT U2OS in response to IR. e High-content imaging quantification comparing the ratio of 53BP1 foci to γ-H2AX foci in both βarr1−/− and WT U2OS cells (*p < 0.05). f βarr1 and 53BP1 were downregulated in U2OS cells obtaining a range of different expression levels of 53BP1. Data are mean ± s.e.m of three independent experiments. g Clonogenic cell survival analysis in U2OS cells revealed a significant difference in survival after exposure to IR in the conditions tested; bar graph for 4 Gy (***p < 0.0001 compare with WT). Data are mean ± s.e.m of an experiment run n = 3. Fractional survival plot as a function of 53BP1 protein expression levels (r = 0.93) in U2OS cells

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