FIG. 8.

MG132 prevents the decrease in H5 staining and its relocalization to splicing speckles. (A) Mock- and HSV-1 WT KOS-infected cells were left untreated (−) or were treated with 50 μM MG132 added 1 h after infection, and nuclear extracts were prepared at 6 h. Samples of the nuclear extracts were fractionated by SDS-PAGE, and Western blot analysis was performed with H5 antibody, specific for phosphoserine-2, and H14 antibody, specific for phosphoserine-5 (P-Ser2). The blots were overexposed to detect the faint, slower-migrating species that are marked by arrows. (B) WT KOS-infected cells were either left untreated (−) or were treated with MG132 added 1 h after infection as indicated. At 6 h after infection, samples of nuclear extracts (NE) were either fractionated directly by SDS-PAGE or were immunoprecipitated (IP) with anti-ubiquitin antibody (left and middle panels) (12). The blot was probed with anti-RNAP II antibodies 8WG16 and ARNA3 to detect all species of RNAP II. The panel in the middle is a darker exposure of the panel on the left. Arrows indicate the position of slower-migrating ubiquitinated forms of RNAP II. In a parallel experiment, after immunoprecipitation with anti-ubiquitin antibody, precipitated proteins bound to the antibody-protein A Sepharose beads were eluted in 1% SDS in phosphate-buffered saline at 60°C for 15 min as previously described (12). The eluate was diluted with 20 volumes of phosphate-buffered saline containing 100 mM NaCl and1% Triton X-100. Samples were then immunoprecipitated with anti-RNAP II antibodies 8WG16 and ARNA3 and were fractioned by SDS-PAGE. Western blot analysis was performed with 8WG16 and ARNA3. (C) WT HSV-1 KOS-infected cells were either left untreated (left panels) or were treated with 50 μM MG132 at 4 h after infection (right panels). Cells were fixed at 8 h and stained with phosphoserine-2 antibody H5, antibody to ICP4, and antibody to SR splicing factor SC35.