Fig. 4: USP35 interacts with and deubiquitinates BRD4.
A Knockdown of USP35 decreased BRD4 protein level in ER+ breast cancer cells (n = 3). B Knockdown of USP35 accelerated BRD4 degradation. MCF-7 and ZR-75-1 cells expressing con-sh and USP35-sh#2 were treated with cycloheximide (CHX, 10 μM) for the indicated times (n = 3). C MG132 rescued the decreased BRD4 level caused by knockdown of USP35. MCF-7 and ZR-75-1 cells with con-sh or USP35-sh#2 were treated with MG132 (10 μM) for 8 h, and then were subjected to western blot analysis (n = 3). D USP35 interacted with BRD4. 293T17 cells were transiently cotransfected with vector or Flag-BRD4 plasmid together with vector or USP35 plasmid. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody beads followed by immunoblotting with the indicated antibodies. E USP35 interacted with BRD4 in ER+ breast cancer cells. MCF-7 and ZR-75-1 cell lysates were subjected to immunoprecipitation with anti-USP35 antibodies and rabbit IgG (negative control). F USP35 promoted BRD4 deubiquitination. 293T17 cells were transiently cotransfected with Flag-BRD4 or HA-ubiquitin plasmid together with USP35WT or USP35C450A plasmid, and then were treated with MG132 (10 μM) for 8 h. Cell lysates were incubated with anti-Flag antibody and immunoblotted with the indicated antibodies. BRD4 levels were quantified and normalized to corresponding β-actin levels, which were presented as fold changes relative to the controls that were set to 1. G, H Immunohistochemistry analysis showed a positive correlation between USP35 and BRD4 protein levels in ER+ breast tumors (n = 18) (H). The representative images are shown on the right (G). Scale bar = 50 μm. All experiments were performed at least three times. Data are shown as Mean ± SEM. **P < 0.01, ***P < 0.001, ns no significance.
