Figure 6. SMARCB1, UL114 and UL44 are associated with the chromatin and the nuclear matrix in HCMV infected fibroblast cells. Mock- and HCMV-infected fibroblast cells harvested at indicated time points were subjected to sub-nuclear fractionation to obtain whole chromatin fraction and core nuclear matrix. Proteins from equal cell equivalents from each fraction were analyzed by western blotting with the indicated antibodies. doi:10.1371/journal.pone.0034119.g006 Nuclear matrix preparation Nuclear matrix proteins were fractionated from the indicated cells according to the method of He and collaborators (1990) [68]. Cells were washed twice in PBS and treated with 500 ml of CSK buffer (10 mM PIPES pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EDTA, 1 mg/ml leupeptin and pepstatin, 1 mM PMSF and 0.1% Triton X-100) for 10 min on ice. The cells were collected and centrifuged at 5000 g for 2 min. The soluble cytoplasmic fraction was removed and the pellet resuspended in 200 ml of CSK buffer containing 100 U RNase-free DNase I (Roche Diagnostics). After 15 min at 37uC, ammonium sulfate was added to a final concentration of 0.25 M. The samples were rotated 5 min at room temperature and centrifuged as above. The soluble chromatin fraction was removed, the pellet washed in CSK buffer with 2 M NaCl for 5 min at 4uC and centrifuged as above. The supernatant was removed and the nuclear matrix pellet resuspended in 100 ml 36 Laemmli buffer and equal cell equivalents from each fraction were subjected to conventional western blot analyses. Immunoprecipitation 750 mg of either mock or HCMV lysates incubated with 2.5 mg of rabbit polyclonal SMARCB1 antibody (a kind gift from Dr. Imbalzano, University of Massachusetts Medical School, US) or 750 mg of HCMV lysate without antibody were incubated for 1 h at 4uC with gentle agitation. Immunocomplexes were precipitated by the addition of Protein G beads (GE Healthcare Bio-Science AB) and incubated for 2 h at 4uC. After sufficient washing with modified RIPA buffer the immunoprecipitate was eluted in 16 Laemmli NuPage buffer by preheating to 70uC for 20 min and separated by 10% Nupage SDS-PAGE and further subjected to conventional western blot analysis. Western blotting Proteins were separated by using the NuPage system (10% gel) (Invitrogen) as described by the manufacturer and electroblotted to a polyvinylidene fluoride membrane (Immobilon-P/Immobilon- FL, Millipore). Primary antibodies used were mouse anti-GST Human Cytomegalovirus UL114 Interact with SMARCB1 ((3D4), sc-57753, Santa Cruz Biotechnology), goat anti-lamin A/C antibody (N-18, Santa Cruz Biotechnology), rabbit anti-histone H1 antibody (FL-219, Santa Cruz Biotechnology), mouse anti- UL57 ((anti-cytomegalovirus ICP8, CH167), ab-53493 Abcam), rabbit anti-UL114 (polyclonal ascites/serum raised against a synthetic peptide comprising amino-acids 11–28+cys of UL114), mouse anti-UL44 (cat. No ABV006, Autogenbioclear), rabbit anti- SMARCB1 (ab12167, Abcam), mouse anti-GAPDH (cat. no. 4300, Ambion), mouse anti-BRG-1 ((G7), sc-17796 Santa Cruz Biotechnology), mouse anti-BAF155 ((DXD7), sc-32763 Santa Cruz Biotechnology) and mouse anti-BAF170 ((E6), sc- 17838 Santa Cruz Biotechnology). Secondary antibodies were peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Promega). Membranes were visualized with the use of ECF-PLUS (Amersham) and PhosphoImager (Molecular Dynamics). Immunofluorescence microscopy Cytospins with 105 cells were fixed with ice-cold methanol/ acetone (1:1) for 90 s and frozen at 270uC until use. Cytospins were washed in TBS before incubation with primary and secondary antibodies diluted in serum diluent (TBS with 12.5% HCMV-negative serum). The cytospins were incubated at 4uC over night with the primary antibodies; mouse monoclonal anti- UL44 (2 mg/ml) (CA006-100, Virusys Corp.), rabbit anti- SMARCB1 (5 mg/ml) (ab12617, Abcam), mouse anti-BAF155 (DXD7) (0,4 mg/ml) (sc-32763 Santa Cruz Biotechnology Inc.), mouse anti-BAF170 (E-6) (0,4 mg/ml) (sc-17838 Santa Cruz Biotechnology Inc.), mouse anti-BRG-1 (G-7) (0,4 mg/ml) (sc- 17796 Santa Cruz Biotechnology Inc.) before incubation with secondary antibodies, anti-rabbit Alexa FluorH 594 f(ab9)2 (Molecular Probes A11072) and anti-mouse Alexa FluorH 488 f(ab9)2 (Molecular Probes A11070), for 1 hour at room tempera- ture. The cover slips were mounted in Vectashield medium containing Dapi (1.5 mg/ml) (Vector Laboratories) and analyzed using a Zeiss LSM 510 META confocal microscope (636/1.4 Plan-Apochromat oil-immersion objective) with the following settings: DAPI; Blue diode 405 laser/band-pass filter 420– 480 nm, Alexa488; Argon–ion 488 laser/long-pass filter 505 nm, Alexa 594; and HeNe 543 laser/long-pass filter 560 nm. Sections (2.5–3 mm thick) were optically sliced (optical slice thickness of ,0.8 um) into 8–10 images that were projected on top of one another to give the images presented. Plasmids The UL114 DNA sequence was PCR-amplified for cloning into the ECORI site of the two-hybrid vector pGBKT7 (MATCH- MAKER two-hybrid systems, Clontech) with primers: 59- CGG AAT TCA TGG CCC TCA AGC AGT GGA TG-39 (forward) and 59- CCC CGA ATT CAC CCA CAG AGT CGC CA-3 (reverse) and pDEST14 –UL114 [26] as a template. All clones were confirmed by sequencing the insert on both strands. Yeast Two-hybrid Screening The Clontech GAL4 MATCHMAKER yeast two-hybrid system was used according to the manufacturer’s (Clontech) instructions. The Pretransformed MATCHMAKER brain cDNA library (PT3183-1) were precloned into a yeast GAL4 activation domain (AD) vector (pACT2, LEU2+), pretransformed into Saccharomyces cerevisiae host strain Y187 (MATa, MEL1, lacZ) and used to screen for binding partners for UL114 cloned into pGBKT7 vector in frame to the DNA binding domain (construct pGBKT7 -UL114) in the yeast strain Saccharomyces cerevisiae host strain AH109 (MATa, HIS3, ADE2, MEL1, lacZ). PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e34119