Ubiquitin enhancement of intracellular degradation of LMP1. (A) Pulse-chase analysis of LMP1-GFP and Ub-LMP1-GFP expression. DG75 cells were transfected with expression constructs LMP1-GFP or Ub-LMP1-GFP, and at 30 hours after transfection, the cells were metabolically labeled for 10 minutes and processed for pulse-chase analysis as described in “Materials and methods.” ^* denotes a truncated Ub-LMP1-GFP protein band. (B) Densitometric analysis of LMP1-GFP and Ub-LMP1-GFP expression. Band intensities were quantified by analysis of the imaging data and plotted as a relative percentage of the signal at time 0 for LMP1-GFP, Ub-LMP1-GFP, and 2 control ubiquitin-LMP1 fusion proteins: Ub-Ala/Gly-LMP1-GFP and Ub-Gly/Met-LMP1-GFP. (C) Effect of the proteasomal inhibitor Lactacystin on the stability of LMP1. Duplicate aliquots of DG75 cells were transfected with expression constructs LMP1-GFP and Ub-LMP1-GFP; 36 hours later, the proteasome inhibitor Lactacystin was added at a final concentration of 10 μg/mL for 12 hours to one of each pair of duplicates. Cell lysates were separated by electrophoresis for immunoblotting with the GFP-specific antibody. The absence (-) or presence (+) of Lactacystin is indicated. Densitometric analysis of the LMP1-GFP and Ub-LMP1-GFP (upper band) expression products are shown.