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Fig. 3 Necdin regulated the stabilization of MYL12A (A and B) Expression of MYL12 A/B/MYL9 in hearts from WT and necdin-deficient (KO) mice. Data are presented as means ± SEM, WT: n = 3; KO: n = 3; ?p < 0.05, ??p < 0.01 and ???p < 0.001, unpaired t test. (C and D) Overexpression of necdin increased the MYL12A protein levels in H9C2 cells. Representative immunoblots (C) and quantification of three independent experiments (D) are shown. Data are presented as means ± SEM, n = 3; ??p < 0.01, unpaired t test. (E and F) RNA interference of necdin decreased MYL12A in H9C2 cells. Representative immunoblots (E) and quantification of three independent experiments (F) are shown. Data are presented as means ± SEM, n = 3; ??p < 0.01, unpaired t test. (G and H) RNA interference of necdin accelerates the degradation of MYL12A protein levels in H9C2 cells. Cycloheximide (CHX, 200 ?g/mL) was used to inhibit protein synthesis. The representative immunoblots (G) and quantification of three independent experiments (H) are shown. Data are presented as means ± SEM; ?p < 0.05, ??p < 0.01, ???p < 0.005, Bonferroni post hoc analysis, two-way ANOVA; NC, negative control. (I) RNA interference of necdin increases the ubiquitination level of MYL12A. H9C2 cells were transfected with the indicated plasmids or siRNAs. Cell lysates were immunoprecipitated with an antibody against Myc, and ubiquitination was detected with an anti-HA antibody.