Coupled in vitro transcription and translation and co- immunoprecipitation The GAL4 activation vector (AD) (pACT2) used for construct- ing pretransformed libraries lacked both a T7 RNA polymerase promoter and an epitope tag. By using PCR with appropriate primers (as indicated by MATCHMAKER Co-IP kit, BD Bioscience) we introduced T7 and HA tag sequences upstream of the collected library cDNA while amplifying the insert for in vitro transcription and translation. In vitro transcription/translation was carried out using the TNTH Quick Coupled transcription/ translation system (Promega Corp.) to synthesis 35S labeled and tagged protein according to the manufacturer’s instructions. Following translation, co-immunoprecipitation according to the manufacturer’s instructions (Matchmaker Co-IP kit, PT3323-1, BD Bioscience) was carried out followed by conventional SDS- PAGE (8% SDS–polyacrylamide) and PhoshorImager (Molecular Dynamics). Expression and purification of recombinant proteins Expression and purification of UL114, His6-UL44 and His6- NEIL1 (hFPG1) were carried out as described previously [26,69]. Human full-length SMARCB1 (UltimateTM ORF Clone IOH29630, pENTRTM221 Invitrogen) which are compatible with the Escherichia coli (E.coli) Expression System Gateway Technology was transferred into pDEST15 (GST-tag at the N- terminal site) by recombination according to the manufacturer’s protocols. E.coli BL21-AITM cells were transformed with pDEST15-SMARCB1 plasmid. Cells were grown at 37uC in 3 L of LBamp medium containing D-Sorbitol and Betaine to an absorbance of approximately 0.3 at 600 nm. Expression was induced by addition of L-arabinose to a final concentration of 0.2%. Cells were grown for an additional 16 hours at 18uC and then centrifuged at 6000 g for 20 min and stored at 220uC before use. The thawed cell pellet was resuspended in 45 mL PSB buffer containing 5 mM DTT, and the cells were lysed by sonication. Next, the lysate was centrifuged at 13000 rpm for 30 min at 4uC. The supernatant was applied directly to a 5 mL GStrap column equilibrated in PBS containing 5 mM DTT and the tagged protein was eluted in 50 mM Tris-HCl, pH 8.0 containing 10 mM reduced glutathione and 10 mM DTT. Fractions containing purified protein were concentrated and further purified by gel filtration chromatography (superdex75 equilibrated in 10 mM Tris-HCl pH 7.5, 50 mM NaCl and 10 mM b-ME). Expression and purification of crude GST extract E.coli BL21 codon Plus cells (BL21-CodonPlus (DE3)-RIL strain, cat. nr. 230245, Stratagene) were transformed with pGEX-3X (Code no: 27-4803-01, Pharmacia Biotech) to express crude GST. The transformed cells were grown at 37uC in 0.5 L LB medium containing 100 mg/ml ampicillin and expression of GST was induced with 1 mM isopropyl-ß-d-thiogalactopyranoside (IPTG) at OD600 of 0.7. The cells were harvested after four hours. The cell pellet was resuspended in PBS with 1 mM DTT (5 ml per g cell pellet) and cell-free extract was prepared by sonication and centrifugation (14000 g) for 30 min. GST pull-down assay Crude GST-extract and purified GST-SMARCB1 were subjected to GST pull-down analysis with the Rapid purification of GST-Fusion Proteins kit (Cat 3 MG-101, Bioclone Inc) using the BcMagHGST magnetic beads according to the manufacturer’s protocol with some modifications. Briefly, 30 ml BcMag beads were incubated with 10 mg purified recombinant GST-SMARCB1 Human Cytomegalovirus UL114 Interact with SMARCB1 or 50 mg crude GST-extract (negative control) for 30 min at room temperature. Next, 20 mg purified recombinant UL114 or 3 mg of modified RIPA lysate from mock or HCMV infected cells harvested at 72 hpi were added to the BcMagHGST magnetic beads. After washing in GST Binding/Washing buffer the co- precipitate was eluted in NuPage loading buffer by heating at 70uC for 20 min. The proteins were separated by SDS-PAGE and verified by western blot analysis. His pull-down assay Purified His6-UL44, His6-NEIL1, GST-SMARCB1 and GST were subjected to conventional his-tag pull-down analysis with DynabeadsH His-Tag Isolation & Pull-down (Cat. No 101.03D) according to the manufacturer’s protocol (Dynal, Oslo, Norway) with some modifications. Briefly, 12.5 ml Dynabeads were incubated with 15 mg His6-UL44 or His6-NEIL1 in binding-buffer (50 mM Na-Phosphate, pH 8.0, 600 mM NaCl, 50 mM Imidaz- ole, 0.01% Tween-20). Next, the Dynabeads were incubated with GST-SMARCB1 (20 mg) or GST (20 mg) in Pull-down buffer ( 6.5 mM Na-phosphate, pH 7.4, 140 mM NaCl, 50 mM Imidaz- ole, 0.01% Tween-20). Finally, the Dynabeads were incubated with 60 ml His Elution buffer (300 mM Imidazole, 50 mM Na- phosphate pH 8.0, 300 mM NaCl, 0.01% Tween-20). The eluted proteins were analyzed by SDS-PAGE. Supporting Information Figure S1 In vitro binding analysis of HA-tagged clone 4 and c-myc-tagged UL114 in 35S-labeled proteins using the TNT coupled transcription/translation system. The proteins were transcribed and translated in vitro with 35S- methionine in the translation mixture to generate radioactive labeled products from vectors pACT2-clone 4 (HA-epitope) and pGBKT7-UL114 (c-myc epitope). The translated clone 4-HA and UL114-c-myc were immunoprecipitated with either anti-HA or anti-c-myc-antibodies, eluted from the Protein G beads and immunoprecipitates (10 ml) were subjected to 8% SDS-PAGE and PhosphoImaging. Lane 1: UL114-c-myc+c-myc antibody. Lane 2: clone 4-HA+HA-antibody. Lane 3: clone 4-HA+UL114-c-my- c+HA-antibody. Lane 4: clone 4+UL114-c-myc+c-myc antibody. (TIF) Figure S2 Mock control for the antibodies UL44 and SMARCB1 used in the co-localization studies of UL44 and SMARCB1 in HCMV-infected fibroblast cells har- vested at 24, 48, and 72 hpi. The cells were fixed and subjected to double-staining for UL44 (mouse Mab-UL44) and SMARCB1 (rabbit Pab-SMARCB1) for immunofluorescence microscopy. Secondary antibodies used for staining were: UL44 in green (anti-mouse 488) and SMARCB1 in red (anti-rabbit 594), and cells were visualized by confocal microscopy. Co-localization was visualized by a merge of the two microscopic determinations, and counterstaining of the nuclei was achieved by the use of DAPI. (TIF) Figure S3 Control for the interaction between SMARCB1 and UL44 using His6-NEIL1 as an irrelevant protein. In vitro pull-down assay of GST-SMARCB1 and His6- NEIL1. Purified GST-SMARCB1 (20 mg) or GST (20 mg) incubated with purified His6-NEIL1 (15 mg) immobilized on magnetic His-tag Dynabeads. Samples were analyzed by SDS- PAGE and Coomassie blue staining. Lane 1: GST-SMARCB1+- Dynabeads His-tag. Lane 2: His6-NEIL1+Dynabeads His-tag. Lane 3: GST-SMARCB1+His6-NEIL1+Dynabeads His-tag. Lane 4: GST+His6-NEIL1+Dynabeads His-tag. Lane 5: GST (input, PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e34119