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. 2022 Feb 22;29(8):1611–1624. doi: 10.1038/s41418-022-00951-y

Fig. 3. PCa-associated SPOP Mutants fail to interact with and promote PrLZ poly-ubiquitination and degradation.

Fig. 3

a A schematic diagram representing the SPOP structural domains and PCa-associated mutations for mapping the interaction domains with PrLZ. b Immunoblot (IB) analysis of WCL and anti-Flag immunoprecipitates (IPs) derived from 293 T cells transfected with Flag-PrLZ, HA-SPOP WT, deletion of MATH domain-SPOP constructs, and BTB domain-SPOP constructs. 30 h post-transfection, cells were treated with 20 μM MG132 for 6 h before harvesting. EV, empty vector. WT, wild type. c IB analysis of WCL derived from 293 T cells transfected with Flag-PrLZ, HA-SPOP WT, and deletion of MATH domain-SPOP constructs. EV, empty vector. WT, wild type. d IB analysis of WCL and anti-HA IPs derived from 293 T cells transfected with Flag-PrLZ, HA-SPOP WT, and PCa-associated SPOP mutants. EV, empty vector. WT, wild type. e IB analysis of WCL derived from C4-2 cells stably expressing HA-tagged SPOP WT or PCa-associated SPOP mutants. EV, empty vector. WT, wild type. f IB analysis of WCL derived from 293 T cells transfected with Flag-PrLZ, HA-SPOP WT, and HA-SPOP F102C mutant. Where indicated, 100 μg/ml CHX was added for the indicated time period before harvesting. EV, empty vector. WT, wild type. g The PrLZ protein abundance in (f) was quantified by ImageJ and plotted as indicated. PrLZ bands were normalized to vinculin. h IB analysis of WCL and Ni-NTA pull-down products derived from PC-3 cells transfected with Flag-PrLZ, HA-SPOP WT, HA-SPOP F102C mutant, and HA-SPOP W131G mutant and His-Ub. Where indicated, 20 μM MG132 was added for 6 h before harvesting the cells. EV, empty vector. WT, wild type. i-k C4-2 cells stably expressing SPOP-F102C or SPOP-W131G mutants were transfected with shPrLZ or shScr and subcutaneously injected into nude mice to establish xenograft model. Statistical analysis of the tumor volumes which were measured every three days and plotted individually (i). Subcutaneous xenograft tumors formed from different groups were dissected (j). Statistical analysis of the weight of the dissected xenografts tumors (k). n = 6 mice per experimental group, the results indicate the mean ± S.D. *P < 0.05. Scr, Scramble. l Representative images of primary PCa patient samples stained for PrLZ expression by immunohistochemistry. Scale bar, upper 200 μm, lower 100 μm. m Mann–Whitney test analysis of PrLZ expression in primary PCa patient samples harboring SPOP-WT or SPOP-mutations.