Figure 5. NPM1-mediated EBNA2 recruitment onto the LMP1 promoter is essential for the maintenance of lymphoblastoid cells.

A). NPM1-depletion, scrambled shRNA expression, or control BJAB-E2 cells were subjected to IP analysis using antibodies for EBNA2, RBP-Jκ, and IgG control, respectively. The expression levels of EBNA2, RBP-Jκ, NPM1, and actin control in each cell line were determined by Western blot. The amounts of EBNA2, BRP-Jκ, and NPM1 contained in each immunoprecipitated protein complex and 2% input of each protein are shown. The immunoblotted images were further quantified using UN-scan. The amounts of the indicated proteins identified in the NPM1 or scrambled shRNA knockdown cell lines are expressed relative to the protein levels observed in the control cells. The relative amount of each protein is shown at the bottom of the image. B–C). The summary of data from A (the first and fourth panels, respectively) is converted as a histogram showing the relative amounts of co-immunoprecipitated RBP-Jκ (B) or EBNA2 (C) from the indicated BJAB-E2 cell lines, which represents the magnitude of EBNA2 and RBP-Jκ association. D). BJAB-E2/LMP1-Luc cells transduced with the lentivirus-expressed NPM1 shRNAs or scrambled shRNA were subjected to a ChIP assay. Immunoblots for the proteins of interest and actin control from each cell line are shown. The promoter occupancy of each protein was quantified by PCR. E). The expression levels of the indicated proteins in each NPM1-depleted or IB4 LCL control were assayed by immune blot. F). IB4 LCL and its derivatives described in (E) were measured for growth over five consecutive days. G). The growth curves represented for the shRNA knockdown and control BJAB cells are shown.