Fig. 2: SPOP interacts with ELK3 for its ubiquitination and degradation.

a, b ELK3 interacts with SPOP. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to determine ELK3 interaction to E3 ligases by IP and WB. c Evaluation of endogenous interaction between ELK3 and SPOP. The cell lysates extracted from HeLa cells treated with MG132 (10 μM) for 8 h were used to evaluate the ELK3 and SPOP interaction by IP and WB. d Confirmation of ELK3 and SPOP interaction. The cell lysates extracted from HEK293T cells transiently transfected with His-ELK3 plasmid were used to confirm the ELK3 and partially purified bacterial His-SPOP by pulldown assay (PD) and WB. e SPOP induces ELK3 destabilization. The cell lysates extracted from HEK293T cells transiently transfected with His-ELK3 and dose-increasing Myc-SPOP plasmids were used to evaluate ELK3 protein level by WB. GFP was used as an internal control for equal transfection. f SPOP accelerates ELK3 destabilization. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) were used to evaluate the ELK3 half-life by WB. g SPOP knockdown increases ELK3 protein levels. The cell lysates extracted from HeLa cells stably expressing sh-mock or sh-SPOP were used to evaluate the ELK3 protein levels by WB. h Depletion of SPOP did not affect ELK3 RNA expression. The levels of ELK3 mRNA extracted from HeLa cells stably expressing sh-mock or sh-SPOP were assessed by quantitative real-time RT-PCR using TaqMan RNA-to-CT 1-step kit. i SPOP knockdown prolongs ELK3 half-life. The cell lysates extracted from PC-3 cells stably expressing sh-mock or sh-SPOP and treated with CHX (10 μg/ml) were used to evaluate ELK3 half-life by WB. j, k SPOP induces ELK3 ubiquitination in cell and in vitro ubiquitination assay system. The cell lysates extracted from 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 (j). In vitro ubiquitination assay was conducted using commercially available E1, E2, and ubiquitin (Ubi) proteins, along with His-tagged ELK3 purified from E. coli and Myc-tagged SPOP isolated from HEK293T cells overexpressing SPOP (k). Graphs in f and i The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. Data, three independent experiments; Significance, *p < 0.05 obtained by Student t test.