Nucleolin Participates in DNA Damage Response Figure 1. Identification of nucleolin as a c-H2AX-interacting protein. (A) phospho-mimic GST-H2AX (H2AXS139E) pulls down several proteins including nucleolin from the nuclear extract of irradiated HeLa cells. Proteins were visualized by silver staining. (B) Pull-downed proteins from the nuclear extract of irradiated or un- irradiated HeLa cells were visualized by Western blot. Nucleolin was precipitated by both H2AX and H2AX (S139E), but NBS1 was precipitated by H2AX (S139E) from irradiated extract only. (C and D) Nucleolin interacts with c-H2AX. Extracts from normal lymphoblastoid cells were immunoprecipitated with anti-nucleolin antibody (C), anti-c- H2AX antibody (D) or normal rabbit IgG, and then the immuno- complexes were detected by Western blot analysis using indicated antibodies. doi:10.1371/journal.pone.0049245.g001 area (Fig. S2A). As nucleolin was identified as an H2AX-binding protein, we examined if H2AX is required for this recruitment. Figure 2. Nucleolin accumulates at DSB damage sites. (A) GFP- nucleolin is confined into nucleolus without DNA damage and diffuses to nucleolpasm after DNA-damaging treatment (CPT 1 mM; etoposide 1 mM) in HeLa cells. (B) GFP-nucleolin is rapidly recruited to DNA damage sites induced by laser micro-irradiation in U2OS cells. Accumulation of GST-nucleolin into irradiated area is indicated by yellow arrowheads. (C) Detection of nucleolin accumulation around DSB damage sites by ChIP. After transfection of I-SceI plasmids in HeLa- DRGFP cells to generate DSBs, the accumulation of c-H2AX, nucleolin, hMRE11 around DSBs were analyzed by ChIP analysis using specific antibodies. doi:10.1371/journal.pone.0049245.g002 H2AX (+/+) MEFs showed rapid recruitment to irradiated area, but H2AX (2/2) MEFs did not display such recruitment (Fig. PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49245