Fig. 3: The Ras-MEK-ERK pathway is activated immediately after hypertrophic stimulation, phosphorylating Bcl-xL at Serine 14 and promoting nuclear translocation of NFAT3.
From: Ser14 phosphorylation of Bcl-xL mediates compensatory cardiac hypertrophy in male mice
a Heat map of differentially expressed genes in the hearts of WT and Bcl-xL-S14A knock-in (KI) mice 9 hs after TAC or sham surgery. n = 3. b Gene set enrichment analysis plots of the FCεR1, ERK and Integrin pathways enriched after TAC compared to sham in WT mice (Supplementary Table 1). NES, normalized enrichment score. GSEA nominal p value is the statistical significance of the enrichment score by using a phenotype-based permutation test with no adjustment. c Immunoblots showing the phosphorylation status of ERK1/2 in the hearts of WT and KI mice after TAC or sham surgery. Immunoblots were repeated at least three times using biologically independent replicates. d Immunoblots showing the phosphorylation status of Bcl-xL (Ser14) in cardiomyocytes after phenylephrine (PE). e, g Relative expression of pBcl-xL (Ser14)/Bcl-xL. Kruskal–Wallis test with vehicle as the control. p values are shown in the figure (n = 5). f Immunoblots showing the effect of a MEK inhibitor (PD0325901) on PE-induced Bcl-xL-Ser14 phosphorylation in cardiomyocytes. h MS/MS spectrum of a doubly charged ion (m/z 646.81) corresponding to the peptide sequence 7ELVVDFLpSYK16 with a phosphorylation modification at S14 in Bcl-xL. The observed y- and b-ion series confirmed the peptide sequence and phosphorylation modification site. i, j Immunoblots showing the nuclear and cytosolic localization of NFAT3 in WT and S14A KI adult mouse cardiomyocytes transduced with adenovirus harboring H-Ras or LacZ (i) and its quantification analysis (j, n = 5). Two-way ANOVA with Tukey’s multiple comparison test. ****p < 0.0001, ns not significant. k Immunoprecipitation assay using α-H-Ras antibody with heart lysates from mice subjected to 9 hs of pressure overload. The numbers indicate the ratio of Bcl-xL (upper arrow) to H-Ras (lower arrow) by densitometric analysis. l FLAG-pull down assay using rat neonatal ventricular cardiomyocytes transduced with adenovirus harboring FLAG-Bcl-xL for two days in the presence of PE or vehicle for 20 mins. Immunoblots were repeated at least three times using independently prepared cardiomyocytes. n represents biologically independent replicates. Data are mean ± SEM.
