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. 2006 Apr;80(7):3660–3665. doi: 10.1128/JVI.80.7.3660-3665.2006

FIG. 2.

FIG. 2.

Effect of Brd4 CTD on BPV1 and mouse Py ori-dependent DNA replication. (A) Southern blot analysis of newly replicated BPV1 ori reporter DNA. C127, CHO, or C33A cells were transfected with BPV1 core ori reporter pUCAlu (C127, 1 μg; CHO, 100 ng; C33A, 250 ng) as well as with pCG expression plasmids (21) for viral replication protein E1 (C127, 2 μg; CHO, 500 ng; C33A, 5 μg) and with either wt E2 or a mutated form that does not bind Brd4 (lanes wtE2 and 37/73, respectively) (C127, 1 μg; CHO, 250 ng; C33A, 1 μg) (23). In addition, either the Brd4 CTD expression plasmid pCGCTD (+) or control vector pCGdXS (−) (C127, 3 μg; CHO, 500 ng; C33A, 3 μg) was cotransfected into the cells. Low-molecular-weight DNA was extracted at days 2 and 3 after transfection of cells by electroporation, digested with HindIII (to linearize the reporter DNA) and DpnI (to digest the unreplicated DNA), and analyzed by Southern blotting using the radioactively labeled probe specific to the ori reporter plasmid. “marker” indicates hybridization control with 100 pg of linearized pUC18 (lane 1) or pUCAlu DNA (lanes 10 and 19). The shorter DpnI fragments of unreplicated plasmid DNA were left out of the gel. (B) Western blotting analysis of the same transfected cells on day 2, using an anti-E2Tag antibody that recognizes both E2 and Brd4 CTD proteins, as indicated on the right; the uppermost band corresponds to nonspecific binding. Equal amounts of total protein were loaded on the gel in each series. “mock” indicates mock-transfected cells; all other lanes are labeled as in panel A. (C) Southern blotting analysis of the newly replicated Py ori reporter DNA in C127 cells. The cells were transfected with 100 ng of Py wild-type or core origin of replication in pUC19 vector (18), 50 ng of Py large T antigen (LTAg) expression vector pCGLT (13), and 3 μg of either the Brd4 CTD expression vector pCGCTD (+) or control vector pCGdXS (−). Low-molecular-weight DNA was extracted on days 2 and 3 after transfection, and Southern blotting was performed essentially as described above for BPV1 transient-replication assays. (D) Parallel Western blotting analysis of the lysates from day 2 of the Py transient-replication assay. Antibodies F4 and F5 (17) were used to detect the LTAg protein. Lanes are labeled as in panel C.