Figure 6.

Marine compounds promote the accumulation of cellular ubiquitin-conjugated proteins. HEK293T cells transiently expressed S5a and ubiquitin proteins by transfection with pcDNA3-HA-S5a and pcDNA3-FLAG-Ub (denoted +). Cells expressing UL76 were transfected with pEF-UL76 (+), and cells not expressing UL76 were instead transfected with the cloning vector, pEF1/Myc-His C (−). Antibodies that recognized the UL76 and FLAG epitopes detected UL76 and ubiquitin-conjugated proteins, respectively. The cellular proteins were resolved in 8.5% SDS-PAGE. An equal level of α-tubulin was used as an internal loading control in each sample. The molecular mass markers are shown on the left in kDa. Cells were treated with the following drug concentrations: MG132 (M), 1 µM; bortezomib (B), 25 nM; compounds 1, 2, 3, and 4, 25 µg/mL. The intensities of the indicated polyubiquitinated area (Ubn, marked by brackets) were quantitated by densitometric analysis. Data were obtained from the average of at least three repeats. The relative ratios of ubiquitinated proteins were normalized to the values with neither UL76 production nor drug treatment. All data points are the averages of at least three repetitive experiments. The error bars indicate standard deviations. The following symbols are used to indicate statistical significance throughout the text: # 0.05 < p < 0.1; * 0.01 < p < 0.05; ** 0.001 < p < 0.01; *** p < 0.001.