Figure 5.

RND3 promoted NICD ubiquitination and protein degradation by facilitating NICD-FBW7 interaction. (A) Noticeable decreases in NICD protein levels were observed along with the elevated RND3 protein levels; this effect was partially attenuated by the treatment with a proteasome inhibitor, MG132. (B) Immunoblot analysis showed that the degradation of endogenous NICD was enhanced by the introduction of RND3 even when protein synthesis was inhibited by the treatment of cycloheximide (CHX). (C) A representative immunoblotting analysis for ubiquitin showed the increase in the ubiquitination level of NICD pull-down complex in the forced expression of RND3 group. Meanwhile, the reduction in NICD protein levels was detected in parallel with this enhanced ubiquitination. (D) With forced expression of RND3, more FBW7-bound NICD protein was detected (Myc-IP section). As a result, less total NICD protein was observed in the same pull-down sample (Input section).