Induction of LMP-1 by IL-10 in Daudi cells. (A) Immunoblot analysis of LMP-1 expression of Daudi cells 48 hours after incubation with 100 ng/mL hIL-10. Total cell extracts of Raji and DG75 cells were used as positive and negative controls, respectively. The numbers on the right side denote the molecular weight in kDa. (B) Immunoblot analysis of LMP-1 expression of hL-10-treated (100 ng/mL) Daudi cells in the presence or absence of IL-10 receptor-blocking antibody (2 μg/mL). (C) EBNA-6, EBNA-1, and LMP-1 immunoblot of Daudi cells treated with 100, 50, 10, or 1 ng/mL hIL-10 for 48 hours. (D) LMP-1 immunoblot of IL-10-treated Daudi cells after the removal of the cytokine. Forty-eight hours after of incubation with 100 ng/mL hIL-10 the cells were washed and replated in the absence of IL-10. Total protein extracts were prepared 24, 48, and 72 hours after the removal of hIL-10 and expression of LMP-1 was monitored by immunoblotting. (E) Total cell extracts of Daudi cells incubated with 10 or 100 ng/mL EBV IL-10 for 48 hours were analyzed for LMP-1 and β-actin expression by SDS-PAGE and immunoblotting. In parallel cultures, Daudi cells were preincubated with 2 μg/mL anti-IL-10R-blocking antibody before the addition of 100 ng/mL cytokine. Of note: because of the low expression of LMP-1, the loading of the cell extracts and the exposure time were optimized, and therefore the immunoblot is not comparable with the other LMP-1 immunoblots.