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. 2013 May 1;8(5):e62481. doi: 10.1371/journal.pone.0062481

Figure 1. RECQ1 interacts with Ku70/80 in vivo.

Figure 1

A. Co-immunoprecipitation analysis of RECQ1 interaction with Ku70/80 using HeLa nuclear extracts. Immunoprecipitations (IP) with antibodies specific for RECQ1, Ku70/80 and preimmune IgG are indicated. Eluted proteins in immunoprecipitate were analyzed by Western blotting and are indicated. RECQ1 IP contained Ku70 and Ku80 subunits but DNA-PKcs was not detected. Reciprocal co-IPs of Ku70/80 also contained RECQ1. B. Association of RECQ1 and Ku70/80 is not mediated via DNA. RECQ1 antibody co-precipitated RECQ1 and Ku70/80 using benzonase-treated extract in IP reaction. C. RECQ1 interacts with Ku in DNA-PKcs deficient and proficient cells. Lysates of MO59J (DNA-PKcs deficient) or MO59K (DNA-PKcs proficient) cells were used for IP using RECQ1 antibody or IgG and analyzed by Western blotting as indicated. D. Immunofluorescence staining of endogenous RECQ1 and Ku70/80. HeLa cells grown on coverslips were either mock-treated or treated with NCS (100 ng/ml, 3 h). Cells were fixed and immunostained using a mouse monoclonal Ku70/80 antibody (1∶200) and a rabbit polyclonal RECQ1 antibody (1∶500). RECQ1 and Ku70/80 were visualized with Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies, respectively, followed by confocal microscopy. Inset shows enlarged portion of the nucleus after NCS treatment; colocalization of RECQ1 (green) and Ku70/80 (red) in cells appears yellow in merged images. In all experiments, input corresponds to 5% of total protein used in IP reactions.