Fig. 5: USP8 deubiquitinates and stabilizes NBR1.

A Exogenous ubiquitination assay of Myc-NBR1 in HEK-293T cells transduced with Flag-USP8 together with Myc-NBR1 and HA-Ub plasmid, and treated with 3-MA for 6 h. B Endogenous ubiquitination assay of NBR1 in DU145 and PC-3 cells treated with 3-MA for 6 h. C In vitro de-ubiquitination assay of ubiquitinated Myc-NBR1 protein with purified Flag-USP8. D Ubiquitination assay of NBR1 in subcutaneous tumors derived from RM-1 cells in high or low PAAG stiffness. E Ubiquitination assay of NBR1 in PCa tissues of Gleason score < 7 or >7. F Ubiquitination assay of Myc-NBR1 in HEK-293T cells co-transfected with Myc-NBR1, Flag-USP8 together with HA-Ub, HA-Ub-K6R, HA-Ub-K11R, HA-Ub-K27R, HA-Ub-K29R, HA-Ub-K33R, HA-Ub-K48R, HA-Ub-K63R, and treated with 3-MA for 6 h. G Ubiquitination assay of Myc-NBR1 in HEK-293T cells co-transfected with HA-Ub, Flag-USP8 together with Myc-NBR1, Myc-NBR1-K767R, Myc-NBR1-K887R, and treated with 3-MA for 6 h. H Stability analysis of NBR1 protein in DU145 and PC-3 cells transfected with shNC or shUSP8, and treated with 40 μM cycloheximide (CHX) for indicated times. I–K RM-1 related stable cells (shNC, shUSP8, and shUSP8+Myc-NBR1) were injected into the flanks of mice, respectively. Tumor volumes were measured every 3 days. Tumor images, growth curves and weight were obtained at day 18 after dissection. L Flow cytometry was used to quantitatively analyze the proportion of CD3⁺ T lymphocytes in CD45⁺ cells, CD8⁺ T lymphocytes in CD45⁺CD3⁺ cells, and the proportion of GZMB⁺ cells or PRF1⁺ cells in CD8⁺ T lymphocytes. The X axis represents the different group. Data in H and K were analyzed by two-way repeated measures ANOVA test. Data are presented as the mean ± SD of at least three independent experiments and were analyzed with one-way ANOVA test unless otherwise stated, *P < 0.05, **P < 0.01, ***P < 0.001.