Figure 5

LMP2A-mediated signaling induces expression of BZLF1. DT40 transfectants stably transfected with a BZLF1 reporter construct (see text for details) were induced with 4-HT to express either wild-type LMP2A (A) or chimeric CD72/LMP2A (B) for the indicated times (lanes 2-8). As control, cells were cultured for 24 h in the presence of anti-IgM antibodies (lanes 1). Subsequently, cleared cellular lysates were subjected to immunoblot analysis with antibodies to BZLF1 or actin (upper and lower panels, respectively). (C) DT40 transfectants were left untreated or induced for 24 h to express either CD72/LMP2A (lanes 1- 2) or phenylalanine mutants Y112F (lanes 3-4) or Y74,85 F (lanes 5-6). BZLF1 protein expression was analyzed as described in A. (D) DT40 transfectants constitutively expressing chimeric CD8/LMP2A, CD8/Igα or CD8/Igβ, respectively, were either left untreated (0) or were stimulated with 10 μg/ml monoclonal anti-CD8 antibody for 24 h. Subsequently expression of BZLF1 and actin was analyzed as in A. Relative molecular mass of a marker protein is indicated on the left of the blots in kDa. Values below anti-BZLF1 blots indicate integrated optical densities of BZLF1 bands normalized to the corresponding actin bands.