Figure 5.

CHOP overexpression promotes BCR-mediated MHV68 lytic gene expression. A, CHOP knockout C-4 cells and control cells (Con) were transfected with vector or CHOP-expressing plasmid with AU1 tag followed by anti-mouse IgG (5 μg/ml) treatment for 48 h at 24 h post-transfection. Immunoblot analyses were performed with the indicated antibodies. The AU1 antibody was used to detect ectopic CHOP expression. Actin was used as a loading control. The molecular mass was marked as indicated (kDa). B, immunofluorescent imaging of MHV68 lytic gene expression in CHOP-expressing C-4 cells. CHOP knockout C-4 cells were transfected with CHOP-IRES-tdTomato–expressing plasmids followed by anti-mouse Ig treatment for 48 h and indirect immunofluorescent staining with specific antibodies against MHV68 lytic antigen (green), CHOP-IRES-tdtomato is shown in red. DNA is stained blue with DAPI. The percentage of MHV68 lytic antigen–positive cells in CHOP-expressing cells and CHOP-negative cells was quantified as shown in the right panel. C, CHOP regulation of RTA promoter activity. M12 cells were co-transfected with Renilla reporter and RTA luciferase promoter (RTA-p) or vector pGL2, together with the CHOP-expressing plasmid with AU1 epitope tag or vector, in the presence (+) or absence (−) of anti-mouse IgG (5 μg/ml) treatment. CHOP expression was detected by AU1 antibody. Luciferase activity was normalized to Renilla activity. Histograms represent mean ± S.D. of triplicate samples (two experiments). A p value of < 0.05 was considered significant.