Figure 1. Mre11 localizes to E2-72kDa containing viral replication foci in E4 mutant infection and it is bound to E4 mutant DNA.

HeLa cells were uninfected (UI) or infected with H5dl1007 (1007) at 30 FFU/cell for the times indicated. (A) and (B) Immunofluorescence staining was performed with antibodies specific for Mre11 (1A, panel a; 1B, panels a, d, g, and j) and the viral E2-72kDa protein (1A, panel b; 1B, panels b, e, h, and k). Bar 10µm. Uninfected cells and cells infected for 12 or 24 h with H5dl1007 were treated with formaldehyde and used for chIP experiments as described in materials and methods. (C) Western blotting was performed to confirm immunoprecipitation of Mre11, E2-72kDa and PI3K with their respective antibodies; representative blots are shown. Lanes from samples that were immunoprecipitated with specific Ab or mock immunoprecipitated are labeled (+) and (−), respectively. (D) PCR amplification using primers specific to the E1b region was performed on chromatin samples prepared from uninfected and H5dl1007 infected cells that were immunoprecipitated (+) with Mre11, PI3K, or E2-72kDa Abs as indicated, or mock immunoprecipitated in parallel without the addition of Ab (−). PCR reactions were fractionated on a 1% agarose gel and stained with ethidium bromide to visualize the 400bp expected PCR product. Total input chromatin (TIC) samples prepared from UI (TIC1) and H5dl1007 infected cells (TIC2) were included to indicate input DNA levels. ChIP experiments with the Mre11 and E2-72kDa antibodies were performed 3 times with similar results. ChIP experiments with Mre11 and PI3K antibodies were performed twice with similar results.