Fig. 5

Rad18 interact with MRE11 and promote formation of MRN complex. A SDS-PAGE separation and silver staining of proteins co-immunoprecipitated with a Rad18-specific antibody. Interacting proteins were subjected to mass spectrometry analysis. B List of MRE11, PCNA and RPA1 picked from interactors of Rad18. C Representative IF images of double staining MRE11 and Rad18. Scale bar =10 μm. D Co-IP showed that MRE11was pulled down by Rad18. E Co-IP showed that HA-tagged MRE11 was pulled down by Flag specific antibody in lysate of 293 T cells which co-transfected with FLAG-tagged Rad18 and HA-tagged MRE11 expression vectors. F and G Protein expression of Rad18 and MRE11in hFOB1.19 and OS cell lines. H Doxorubicin treatment increased interaction between Rad18 and MRE11. Total lysates derived from 143B cells treated with 0 μM or 5 μM doxorubicin were immunoprecipitated with Rad18 antibodies. The precipitates were probed with the indicated antibodies. I Rad18 knockout decreased MRN complex formation. Total lysates derived from 143B cells treated with 5 μM doxorubicin for 0 h and 6 h were immunoprecipitated with Rad18 antibodies. The precipitates were probed with the indicated antibodies. J Western-blotting analysis showed Rad18 knockout inhibited doxorubicin induced ATM phosphorylation. K Immunofluorescence staining of 143B-sgNC and 143B-sgRad18 cells with p-ATM antibodies after the cells were infected with lenti-sgNC or lenti-sgRad18 and subsequently treated with doxorubicin in time gradient. Scale bar =10 μm. L Schematic diagram of the mechanism that Rad18 promotes HR pathway through interaction with MRE11