Fig. 3. Accumulation of UBB⁺¹ is reversible after shutting off expression. Flow cytometric analysis of the percentage of UBB⁺¹ (A) or UBB⁺¹/GFP (B) positive cells at 0 hours, 12 hours and 36 hours after shutting down maximal UBB⁺¹ expression. Ub^G76V-GFP HeLa cells were not transfected (control), transfected with empty pcDNA3 vector (e) or transfected with the UBB⁺¹ Tet-off vectors. After 64 hours of maximal expression (absence of Dox) a baseline sample was taken (0 hours). UBB⁺¹ expression was continued at maximal levels (Dox–) or shut down by addition of 10 ng/ml Dox (Dox+) and samples were analyzed after 12 hours and 36 hours of Dox treatment. Results are the means ± s.e.m. of two or three independent duplicate experiments, ^*P<0.01; #p=0.099 ANOVA between groups indicated at 36 hours.