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. 2018 Sep 24;7(9):75. doi: 10.1038/s41389-018-0086-y

Fig. 7. USP14 and UCHL5 inhibitors increase the ubiquitination of ERα and decrease ERα-mediated transcription activity.

Fig. 7

a Total RNAs were collected from T47D cells exposed to b-AP15 or PtPT for 12 h and subjected to RT-qPCR analysis for ERα and ERα-targeted gene, PS2 mRNA expression. Mean ± SD (n = 3). *p < 0.05, #p < 0.01. b T47D cells were treated with cycloheximide (CHX) for 0, 6, 12, 24 h and co-treatment of CHX and b-AP15 or PtPT at the same time. Protein lysates were collected and subjected to western blot assay for ERα protein level. Representative images were shown. c Relative quantifications of ERα band density were calculated. *p < 0.05. d The total proteins were collected from T47D cells treated with b-AP15 (1 μM) or PtPT (5 μM) for 24 h and MG132 (10 μM) for 6 h before it was collected, immunoprecipitated with ERα antibody beads and immunoblotted for ERα and ubiquitin (Ub). e T47D cells were transfected with luciferase reporter plasmid containing estrogen receptor elements (EREs). After transfection for 24 h, cells were exposed to b-AP15 and PtPT for 24 h. Protein lysates were collected and dual-luciferase assay was used to measure relative luciferase activity. Three independent experiments were performed.*p < 0.05