Figure 7(A) Representative immunoblot and its quantification of protein levels of neddylated and unnedylated Cul1 in NRCMs after Cand2 overexpression. Analysed by t-test, n=3 biological replicates per conditions. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001 (B) Cul1 neddylation analyzed by Western blot in NRCMs depleted of Cand2. NRCMs were treated with MLN4924 (100 nM) to block Cul1 neddylation. Quantification of neddylated and unnedylated Cul1 protein levels after MLN4924 treatment for 24h. t-test, n=4 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001 (C) Cell size area measurement of NRCMs after Cand2 KD and neddylation inhibition (MLN). Analyzed by One-way ANOVA. n=3 independent experiments with >150 cells. Violin plot: the middle horizontal lane - median, upper lane - 3rd quartile, lower lane - 1st quartile. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001 (D) Grk5 expression in NRCMs upon Cand2 KD and Cul1 neddylation inhibition (MLN) analysed by immunoblot and RT-qPCR. Analyzed by One-way ANOVA, n=4-5 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001 (E) The half-life of Grk5 in HeLa cells overexpressing Cand2 measured by cycloheximide chase. n=2 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001 (F) Representative immunoblot and quantification of Grk5 protein levels as well as quantification of mRNA levels after Cul1 KD. t-test, n=4 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001 (G). Immunoblot after Cand2 KD and Grk5 OE. Cell surface area measurement of NRCM for indicated groups. Analyzed by One-way ANOVA. n=3 independent experiments with >150 cells. The middle horizontal lane on violin plot - median, upper lane - 3rd quartile, lower - 1st quartile. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001 (H) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs for indicated groups. Analyzed by One-way ANOVA, n= 6 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001 (I) Immunoblot after Cand2 OE and Grk5 knockdown. Cell surface area measurement of NRCM for indicated groups. Analyzed by One-way ANOVA. n=3 independent experiments with >150 cells. The middle horizontal lane on violin plot - median, upper lane - 3rd quartile, lower - 1st quartile. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001 (J) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs for indicated groups. Analyzed by One-way ANOVA. n=6 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001