Fig. 7: NUSAP1 stabilizes Notch2 expression by binding to N2ICD through the R422 site.
From: PRMT1-catalyzed NUSAP1 methylation enhances Notch2 signaling and 5-FU resistance in gastric cancer
A Western blot analysis showing the effects of NUSAP1 knockdown on Notch2 and its downstream signaling proteins (C-Myc, CyclinD3 and CDKN1A) in BGC-823-5-FU-R and SGC-7901-5-FU-R cells. Tubulin was used as a loading control. B Western blot analysis of NUSAP1 and Notch2 protein levels in HEK293FT cells treated with NH4Cl or MG132, with or without NUSAP1 knockdown. C Co-IP assay in BGC-823-5-FU-R and SGC-7901-5-FU-R cells showing that NUSAP1 knockdown promotes Notch2 ubiquitination. HA-tagged ubiquitin (HA-Ub) was used to detect ubiquitinated Notch2 in the presence of MG132. D Western blot analysis of Notch2 and its downstream signaling proteins in cells expressing MYC-tagged wild-type (WT) or mutant (R418K, R422K) NUSAP1, demonstrating the importance of R422 methylation for regulating Notch2 signaling. E Co-IP assay showing that WT NUSAP1 and R418K NUSAP1, but not the R422K mutant, inhibits ubiquitination of Flag-tagged N2ICD in HEK293FT cells treated with MG132. F, G Cycloheximide (CHX) chase assay to determine the half-life of Notch2 in HEK293FT cells expressing WT or mutant (R418K, R422K) NUSAP1. Western blot analysis of Notch2 protein levels over time is shown, with degradation kinetics plotted in (G). H Co-IP assay showing the binding between MYC-NUSAP1 WT, R418K, R422K and Flag-N2ICD in HEK293FT cells. I Western blot analysis using the NUSAP1-R422me2-specific antibody showing that R422 methylation enables NUSAP1 to bind to Flag-tagged N2ICD in BGC-823-5-FU-R and SGC-7901-5-FU-R cells. J Western blot analysis showing the effects of PRMT1 inhibitors (AMI-1 and DCLX069) and PRMT1 down-regulation on the interaction between MYC-NUSAP1 and Flag-N2ICD.
