Fig. 3: Cancer-derived SPOP mutations fail to promote ELK3 degradation.

a ELK3 interacted with MATH domain of SPOP. Upper panel Schematic diagram of SPOP domains. The numbers at the MATH domain indicates SPOP mutations observed in PCa patients. Bottom panels The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated MG132 (10 μM) for 4 h were used to determine the SPOP’s binding domain by IP and WB. b MATH domain deletion of SPOP suppressed ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated MG132 (10 μM) for 4 h were used to evaluate the SPOP-dependent ELK3 ubiquitination by IP and WB. c The deletion of the MATH or BTB domain of SPOP abolishes ELK3 destabilization. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were used to evaluate ELK3 protein levels by WB. GFP was used as an internal control for equal transfection. d MATH domain mutation of SPOP abrogates interaction with ELK3. The cell lysates of HEK293T cells transiently transfected with the indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between SPOP and ELK3 by IP and WB. e MATH-F133V mutation of SPOP critically affects ELK3 stability. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) for indicated period were used to evaluate ELK3 protein levels by WB. The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. GFP was used an internal control for equal transfection. Data, three independent experiments; Significance, *p < 0.05 obtained by Student t test. f SPOP mutations at MATH domain inhibits endogenous ELK3 destabilization. The cell lysates of 22Rv1 cells stably expressing SPOP-wt or each of SPOP mutants (Y87C, W131G, and F133V) were used to evaluate ELK3 protein levels by WB. g SPOP mutations at MATH domain abolishes ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate ELK3 ubiquitination by IP and WB.