Fig. 4: Phosphoproteomic screening of Clk4-cKO hearts identifies NEXN as a potential target. | Nature Communications

Fig. 4: Phosphoproteomic screening of Clk4-cKO hearts identifies NEXN as a potential target.

From: CDC-like kinase 4 deficiency contributes to pathological cardiac hypertrophy by modulating NEXN phosphorylation

Fig. 4

a Volcano plots showing changes in phosphopeptides in Clk4-cKO mice compared with MCM mice, as identified using mass spectrometry (MS). The red dots are peptides with fold-changes (Clk4-cKO/MCM ratios) > 1.2 (P < 0.05, t test), and the blue dots are those with fold-changes < 1/1.2 (P < 0.05, t test). In, increase; De, decrease. b Hierarchical clustering analysis of the specified phosphoproteome. A total of 366 phosphoforms met the aforementioned two criteria. c A table showing the top downregulated phosphopeptides and corresponding phosphoproteins obtained from differential gene expression analysis in Clk4-cKO hearts. d qPCR analysis of cardiac hypertrophic-related genes, including Nppa and Nppb, in NRVMs transfected with siRNAs targeting Pdha1, Snw1,Dusp27, Zc3hc1, Ndrg1, and Nexn; here, Clk4 siRNA was used as a positive control. e–g Coimmunoprecipitation of CLK4 with NEXN in NRVMs transfected with CLK4-flag and NEXN-myc (e, f), and in vivo myocardium infected with AAV NEXN-WT (g). The input represents 6% of the whole-cell lysate used for each immunoprecipitation. IgG was used as a negative control. h Representative western blots showing that CLK4 increased the serine phosphorylation level of NEXN in NRVMs. Samples incubated with calf intestinal phosphatase (CIP) served as a negative control. i Typical western blots illustrating that CLK4 phosphorylated the wild-type NEXN fragment (amino acids 295–671) but not the mutant type (S437A) in a cell-free system. j Western blot detection of the phosphorylated NEXN in Clk4-cKO mouse myocardia using a Phos-tagged acrylamide gel. For a, b, n = 3 animals per group; for d, n = 3 biologically independent samples per group. For a, statistical analysis was performed using unpaired, two-tailed Student’s t test; for d, statistical analysis was performed using one-way ANOVA and Dunnett multiple comparisons test. Data are presented as the means ± S.E.M.; P values are shown in the graph. Source data are provided as a Source Data file.

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