Research Paper Volume 14, Issue 18 pp 7527—7546

Figure 3. FAT10 interacting with USP7 and stabilising USP7 expression. (A) A partial list of FAT10-associated proteins were indicated by immunoprecipitation-mass spectrometry. (B) Protein levels of FAT10 and USP7 in FAT10-overexpressing or FAT10-silenced HK-2 cells were detected by western blotting. Tubulin was used as a loading control. (C) Determination (left) and quantification (right) of FAT10 and USP7 protein levels in HK-2 cells or FAT10-silenced HK-2 cells following treatment with hypoxia or without hypoxia. ^*P < 0.05, ^**P < 0.01. (D) Western blot showing USP7 protein levels in HK-2 cells following treatment with 10 μM MG132 at different times. (E) HK-2 cells transduced with shFAT10 or Flag-FAT10 were treated with MG132. Cells were collected at 6 h and immunoblotted with the antibodies indicated. (F and G) Representative (F) and quantitative (G) results of USP7 protein level in FAT10-overexpression or FAT10-silencing cells. The cells were treated with cycloheximide (CHX, 100 μg/ml) for indicated time points were subjected to western blot analysis. The degradation rate of USP7 protein was calculated according to the ratio of USP7/tubulin. The quantification data represent mean ± SD from three independent experiments and were statistically analyzed with Student’s t-test, ^*P < 0.05, ^**P < 0.01. (H and I) Competitive binding of USP7 was analyzed in a GST-pull down experiment. HEK-293 T cells were transfected with the indicated constructs and lysed for IP using anti-His beads to detect GST binding. (J) Knockdown or exogenous expression of FAT10 in HK-2 cells altered the ubiquitination of USP7. The cells in each group were treated with MG132. (K) Western blotting showing the protein expression of FAT10 and USP7 in FAT10^+/+ RTECs and FAT10^−/− RTECs following treatment with hypoxia or without hypoxia. (L) Ubiquitinated USP7 in in FAT10^+/+ RTECs and FAT10^−/− RTECs following treatment with hypoxia or without hypoxia. The cells in each group were treated with MG132.