Figure 5. Nedd4 interacts with Mecp2 and affects quiescence exit by facilitating Mecp2 degradation.

(A) Reciprocal immunoprecipitation-western blotting (IP-WB) analysis to validate the interaction between endogenous Mecp2 and Nedd4. (B) Co-IP of Mecp2, ubiquitin, and Nedd4 in quiescent 3T3 cells during serum restimulation (SR)-induced quiescence exit. (C) Real-time PCR showing siRNA-mediated Nedd4 knockdown (KD) in 3T3 cells upon SR-induced quiescence exit. Data are presented as means ± SEM; n = 5. ****p<0.0001 by two-way ANOVA. (D–F) The effect of Nedd4 KD on quiescent exit in 3T3 cells determined by WB (D), immunofluorescence (IF) staining of Ki67 and Nedd4 (E), and Ki67/PI staining followed by flow cytometry (F) at the indicated time points. Lower panel in (F): quantification of the percentage of 3T3 cells in the G0 phase. Data are presented as means ± SEM; n = 3. n.s., not significant; ***p<0.001, ****p<0.0001 by two-way ANOVA. (G) Real-time PCR showing Nedd4 overexpression (OE) in 3T3 cells upon SR-induced quiescence exit. Data are presented as means ± SEM; n = 5. ****p<0.0001 by two-way ANOVA. (H–J) The effect of Nedd4 OE on quiescent exit in 3T3 cells determined by WB (H), IF staining of Ki67 and Nedd4 (I), and Ki67/PI staining (J) at the indicated time points. Data are presented as means ± SEM; n = 3. n.s., not significant; ****p<0.0001 by two-way ANOVA.

Figure 5—figure supplement 1. Nedd4 interacts with Mecp2.

(A) Western blotting (WB) of Mecp2 in quiescent 3T3 cells treated with or without MG132 after being released from G0 by serum restimulation (SR). (B) WB of Mecp2 in quiescent 3T3 cells treated with or without chloroquine after being released from G0 by SR. (C) Coomassie staining of Mecp2 co-IP proteins separated by SDS-PAGE. Right: representative peptides of Nedd4 identified by mass spectrometry. (D) WB showing protein levels of Nedd4 in mouse livers before and 6 hr after partial hepatectomy (PHx).