Human Cytomegalovirus UL114 Interact with SMARCB1 Figure 5. Interaction of SMARCB1 and UL44 in HCMV-infected fibroblast cells and with recombinant proteins. (A) Co- immunoprecipitation of endogenous SMARCB1 and UL44 in HCMV-infected fibroblast cells. Equal numbers of mock infected and HCMV infected fibroblast cells were lysed at 72 hpi, and the extracts were immunoprecipitated with anti-SMARCB1. Co-immunoprecipitated proteins were resolved electrophoretically and subjected to immunoblot analysis with anti-UL44 and anti-SMARCB1. Lane 1: Mock lysate immunoprecipitated with anti- SMARCB1. Lane 2: HCMV lysate immunoprecipitated with anti-SMARCB1. Lane 3: HCMV lysate immunoprecipitated with no antibody. Lanes 4 and 5, SMARCB1 and UL44 input in extracts (7 mg, 1% of the total in Lane 4 and 35 mg, 5% of the total in Lane 5). IgGHc: IgG heavy chain. (B) In vitro pull- down assay of GST-SMARCB1 and His6-UL44. Purified GST-SMARCB1 or GST incubated with purified His6-UL44 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- UL44+Dynabeads His-tag. Lane 3: GST-SMARCB1+His6-UL44+Dynabeads His-tag. Lane 4: His6-UL44 (2 mg, 10% of input). Lane 5: GST-SMARCB1 (2 mg, 10% of input). Lane 6: GST+His6-UL44+Dynabeads His-tag. Lane 7: GST (2 mg, 10% of input). Note that spontaneous cleavage of GST occurred in the GST-SMARCB1 protein sample (Lane 5). (……) indicates that samples were run on the same gel. doi:10.1371/journal.pone.0034119.g005 At different time-points post infections, the cells were scraped off for preparation of cell lysates or trypsinized for preparation of nuclear extract or cytospins for immunostaining. Preparation of cell lysates and nuclear extracts Cells for preparation of cell lysates were harvested by scraping cells into ice-cold PBS, followed by centrifugation at 10006 g for 4 minutes at 4uC. Cell pellets were lysed using a modified RIPA buffer (50 mM Tris-HCL, pH 7,4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 (Igepal CA-630, Sigma-Aldrich), 1 mM PMSF and Protease Inhibitor Cocktail (PIC) (P 8340, Sigma-Aldrich), and incubated at 4uC for 15 minutes, followed by centrifugation at 140006g for 15 minutes at 4uC . The supernatants were decanted into a fresh tube and stored at 270uC until used. Nuclear extracts were made by plasmolysis of mock or HCMV-infected (5 PFU/ cell) fibroblast as previously described [66,67]. PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e34119