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. 2021 Aug 28;10(9):2229. doi: 10.3390/cells10092229

Figure 8.

Figure 8

High level of αSyn decreases the pool of ubiquitinated substrates upon downregulation of Tet-RPN11. (A) Western blot analysis of Tet-RPN11 mutant strains treated with cycloheximide. Strains harboring one (1×) or three (3×) gene copies of αSyn or an empty vector (EV) as a control were grown overnight in a galactose medium to induce αSyn expression. The Tet promoter was downregulated by addition of 10 µg/mL doxycycline to the growth medium. The next day, cells were treated with 50 µg/mL cycloheximide to stop de novo protein synthesis. Immunoblotting analysis was performed at the indicated time points after addition of cycloheximide with ubiquitin (Ubi) or αSyn antibodies. GAPDH was used as a loading control. (B) Densitometric analysis of the immunodetection of the ubiquitin conjugates relative to the GAPDH loading control. The ubiquitin/GAPDH ratio was normalized to the ratio of EV (Tet-ON) at 0 h. The significance of the differences was calculated with a t-test (*, p < 0.05; **, p < 0.01, n = 3). (C) Densitometric analysis of the immunodetection of αSyn relative to the GAPDH loading control. The αSyn/GAPDH band ratio was normalized to the corresponding ratio at 0 h and presented as percentage. Values represent the mean ± SEM of three independent experiments. (D) Recovery of the depleted ubiquitin pool is accompanied by αSyn degradation. Western blot of Tet-RPN11 mutant strains after GAL1 promoter shut-off. Cells were grown overnight in a galactose medium to induce αSyn expression and transferred to a glucose medium that repress the GAL1 promoter. Immunoblot analysis was performed at the indicated time points after the shift to a glucose medium with ubiquitin, αSyn and GAPDH antibody as a loading control. (E) Densitometric analysis of the immunodetection of ubiquitin conjugates relative to the GAPDH loading control. The ubiquitin/GAPDH ratio was normalized to the corresponding ratio of EV-Dox at 0 h. The significance of the differences was calculated with a t-test (*, p < 0.05; **, p < 0.01, n = 3). (F) Densitometric analysis of the immunodetection of αSyn relative to the GAPDH loading control. The αSyn/GAPDH band ratio was normalized to the corresponding ratio at 0 h and presented as a percentage. Values represent the mean ± SEM of three independent experiments.