Figure 1.

LMP1 suppresses the expression and transcriptional activity of Foxo3a. (A) HEK-293 cells were transfected with increasing amount of the LMP1 vector (pSG5-LMP1) as indicated. Forty-eight hours post-transfection, cells were harvested for immunoblotting analysis. For the detection of Id1 and Foxo3a, cells were cultured in serum free medium for 6 hrs before harvesting. (B) Total cell lysates of NP69-pLNSX control and NP69-LMP1 cells were analyzed by immunoblotting. For detection of cytoplasmic and nuclear proteins, cells were treated with protease inhibitor, MG132 (20μM) for 4 hrs prior to harvesting. Relative protein expression was calculated using densitometry with the control set at 1. (C) HEK-293 cells were transfected with various doses of the LMP1 expression vector (pSG5-LMP1) and reporter constructs for p27^kip or Bim. (D) HEK-293 cells were transfected with various doses of GFP-Foxo3a and the p27^kip or Bim promoter reporter constructs, together with 40 ng LMP1 expression vector (pSG5-LMP1) or control empty vector (pSG5). Cells were harvested for luciferase reporter analysis 48 hrs post-transfection. Luciferase activity was normalized to β-gal activity. Data shown are the mean ± s.d. of three separate experiments. The relative luciferase unit (RLU) is plotted relative to that of the reporter alone (set at 1).