Fig. 7

USP7 bound to KPNB1 and stabilized KPNB1 expression by deubiquitination. A. UbiBrowser identified deubiquitinases (DUBs) that might interact with KPNB1. B. Western blot analysis showing the reciprocal co-IP in U87MG and U251MG cells demonstrating the binding of endogenously expressed KPNB1 to USP7. C. U87MG and U251MG cells were infected with shUSP7#1 and shUSP7#2. Cells were collected for Western blot analysis and RT-qPCR. Data presented as the mean ± SD of three independent experiments, ns: not significant, one-way ANOVA. D. U87MG and U251MG cells were treated with or without USP7 inhibitor P5091 (10 μM) for 48 h. Then, cells were harvested for RT-qPCR and Western blotting analysis. Data presented as the mean ± SD of three independent experiments, ns: not significant, one-way ANOVA. E. U87MG and U251MG cells were infected with USP7 plasmids. Cells were collected for Western blot analysis and RT-qPCR. Data presented as the mean ± SD of three independent experiments, ns: not significant, one-way ANOVA. F. U251MG cells were infected with shUSP7#1 and shUSP7#2. After 48 h or 72 h, the corresponding groups were treated with MG132 for another 12 h. All cells were harvested for Western blotting analysis. G. U251MG cells were treated with or without USP7 inhibitor P5091 (10 μM) for 48 h then treated with MG132 for another 12 h. All cells were harvested for Western blotting analysis. H-J. U251MG cells were infected with shUSP7 or Myc USP7 (H) or treated with P5091 (I). Cells were treated with cycloheximide (CHX), and all cells were collected for Western blotting analysis at different time points. The half-life of KPNB1 protein is shown (J). K-L. U251MG cells were infected with shUSP7 (K) or Myc USP7 (L) and treated with MG132 for 12 h. Then, cells were collected for Western blot analysis. M-N. GBM tissue microarrays were stained with antibodies against USP7 and KPNB1. The correlation of these two proteins is shown in panel (N). Spearman correlation was used to determine statistical significance; P < 0.001