Fig. 1: REN binds and ubiquitylates SALL4 promoting its proteasome-mediated degradation.

A HEK293T cells were transfected with plasmids encoding for Flag-HA epitope tagged REN/KCTD11 or an empty vector (EV). B Cells were treated with the proteasome inhibitor MG132 and lysed. Whole cell extracts were immunoprecipitated with anti-Flag resin and eluted with Flag peptide. A second immunoprecipitation was carried out with an anti-HA antibody, which was followed by three sequential elutions with 1% SDS, as indicated. Immunocomplexes were resolved by SDS-PAGE. The gel was then stained by silver stain for protein visualization. Co-IPs of C Flag-REN and endogenous SALL4, or D Flag-REN WT or its mutants (ΔBTB, BTB) and exogenous SALL4 from HEK293Ts transiently transfected with indicated plasmids. IPs of E HA-SALL4 or F endogenous SALL4 from HEK293Ts transiently transfected with indicated plasmids. G IP of HA-SALL4 from HEK293Ts transiently transfected with indicated plasmids and treated with MG132 (50 μM, 4 h) to block proteasome, or DMSO as control. In E, F, and G anti-Flag antibody was used to detect the SALL4 polyubiquitylated forms; anti-HA or anti-SALL4 antibodies were used to re-probe blots to assess the levels of immunoprecipitated protein. Total protein lysates are shown in the Input. H, K Representative immunoblotting and I, L densitometric analysis of SALL4 protein levels in Med1-MB cells transiently transfected with indicated plasmids, and treated in K with CHX (100 μg/mL) up to 8 h to block protein synthesis. J qRT-PCR analysis of SALL4 expression in Med1-MB cells overexpressing increasing amounts of HA-REN. Representative immunoblotting of n = 3 biological replicas with similar results are shown in C–H, and K. Densitometric analysis in I and L, normalized on endogenous actin, represent the mean of n = 3 independent experiments ± SD. Data in J are normalized to endogenous Gapdh and Hprt control, expressed as the fold change (FC) versus the control sample value, and represent the mean of n = 3 independent experiments ± SD. *p < 0.05 calculated with two-sided Student’s t-test.