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Fig. 2. High levels of UBB⁺¹ inhibit the proteasome. Flow cytometric analysis of Ub^G76V-GFP HeLa cells for GFP fluorescence as an indication of UPS inhibition (% of cells that are GFP positive). (A) Cells were transiently transfected with empty pcDNA3 vector (control), CMV-Ub wild-type pcDNA3 (Ubwt), CMV-UBB^+1K29,48R pcDNA3 (+1^K29,48R) or CMV-UBB⁺¹ pcDNA3 (UBB+1) or treated with 100 nM epoxomicin (epox) or 1 µM MG132. Significant accumulation of GFP compared with empty vector control is marked with an asterisk (^*P<0.005, ANOVA Bonferroni). (B) Ub^G76V-GFP cells were transiently transfected with the inducible Tet-off UBB⁺¹ expression system and treated with decreasing concentrations of Dox. Significant increase in the percentage of GFP-positive cells compared with levels in cells treated with 1000 ng/ml Dox is marked by an asterisk (^*P<0.05, ANOVA Bonferroni). Results are the means ± s.e.m. of three or four independent duplicate experiments. (C) Representative flow cytometric scatter plots of UBB⁺¹ Tet-off transfected cells treated with 1000, 0.01, 0.0001 and 0 ng/ml Dox as shown in B. GFP-positive cells were detected in the region set as R1. The mean GFP fluorescence and the percentage of cells that are GFP-positive are indicated on the right.

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