Fig. 5

The Cullin 3 SPOP E3 ubiquitin ligase negatively regulates the stability of HnRNPK. A Expression of HnRNPK and SPOP in PrCa cells transfected with miR-206 and miR-613 mimics was detected by Immunoblot analysis. B Relative SPOP mRNA expression levels in PrCa tissues and adjacent normal prostate tissues in a public data set (GSE60329). C Effect of the SPOP expression level on Biochemical recurrence in 36 prostate cancer patients who did not undergo androgen deprivation therapy, chemotherapy, radiotherapy or other anticancer treatment was analyzed, and Kaplan–Meier plots were generated using a Kaplan–Meier Plotter. D WB analysis of WCL and immunoprecipitates (IP) derived from 293 cells transfected with Flag-HnRNPK and various Myc-tagged Cullin constructs. 30 h post-transfection, cells were treated with 10 μM MG132 for 10 h before harvesting. E WB analysis of WCL derived from C4-2 and 22Rv1 (F) cells infected with the indicated lentiviral shRNAs. Infected cells were selected with 1 μg/ml puromycin for 72 h to eliminate non-infected cells before harvesting. G WB analysis of WCL and IP derived from 293 cells transfected with HA-HnRNPK and Flag-tagged BTB domain-containing protein constructs. 30 h post-transfection, cells were treated with 10 μM MG132 for 10 h before harvesting. EV, empty vector. H WB analysis of WCL derived from C4-2 or 293 I cells transfected with increasing doses (0.5–3 μg) of indicated plasmids. Where indicated, 10 µM MG132 was added for 10 h before harvesting. J WB analysis of WCL derived from C4-2 cells infected with the indicated lentiviral shRNAs. Infected cells were selected with 1 μg/ml puromycin for 72 h to eliminate non-infected cells before harvesting. K–N IB analysis of WCL derived from C4-2, 22Rv1, DU145 and mouse embryonic fibroblasts (MEFs) cells with SPOP knockout by the CRISPR technology. O WB analysis of WCLs and His pull-down products derived from 293 cells transfected with indicated constructs and treated with MG132 (10 μM) 10 h. P SPOP knockout cells (sg SPOP) as well as parental C4-2 cells (Con) were treated with 100 μg/ml cycloheximide (CHX), and cells were harvested at the indicated time points. Relative HnRNPK protein abundance was quantified by Image J and plotted in Q. Data was shown as mean ± SD for three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001