Fig. 5: RNF121 directly binds MYCN protein and increases its stability.

a, b Representative immunoblots from cycloheximide (CHX) chase experiments 48 h after RNF121 knockdown using control siRNA or RNF121-specific siRNAs #1 or #4 in SK-N-BE(2)-C (a) and Kelly (b) cells. Cells were then treated with 100 µg/ml CHX for up to 30 min followed by immunoblotting of total cell lysates with anti-RNF121 or anti-MYCN antibodies. The ratio of MYCN protein/Actin protein was artificially set at 1.0 for control samples and then the half-life of MYCN was calculated from the line graph. c RT-PCR of MYCN mRNA expression levels following 48 h of siRNA knockdown of RNF121. d RT-PCR of MYCN and RNF121 mRNA expression levels following 24 h of Doxycyline (Dox) treatment of Dox-inducible MYCN knockdown neuroblastoma cell line, SHEP-Tet21N. e Schematic of PCR primer sites in the RNF121 gene promoter used for ChIP-PCR, detailing the MYCN peak binding summit (−136 bp) and an upstream negative control site (−2285 bp). f ChIP-PCR assays assessing fold enrichment of MYCN protein binding at two sites within the RNF121 gene promoter: the MYCN protein binding summit (−136 bp [Promoter]) or the negative control region (−2285 bp [Control]) using an anti-IgG (control) or anti-MYCN antibody in total cell lysates from either SHEP-TET21N or SK-N-BE(2)-C neuroblastoma cells, both with basal high MYCN expression. g Representative Western blot analysis for endogenous RNF121 after immunoprecipitation of endogenous MYCN in total cell lysates from MYCN amplified Kelly and SK-N-BE(2)-C cells. 5% of the cell lysate was loaded for input.