Fig. 6: Increases in the expression of UBC9 and SUMO2/3 promotes the GPR120 SUMOylation in AMPKα2−/− VSMCs by activating the C-MYC S67 phosphorylation. | Nature Communications

Fig. 6: Increases in the expression of UBC9 and SUMO2/3 promotes the GPR120 SUMOylation in AMPKα2−/− VSMCs by activating the C-MYC S67 phosphorylation.

From: AMPKα2 controls the anti-atherosclerotic effects of fish oils by modulating the SUMOylation of GPR120

Fig. 6: Increases in the expression of UBC9 and SUMO2/3 promotes the GPR120 SUMOylation in AMPKα2−/− VSMCs by activating the C-MYC S67 phosphorylation.The alternative text for this image may have been generated using AI.

A, B Western blotting and quantification of UBC9 and GPR120 expression in cytoplasmic and membrane fractions from AMPKα2−/− VSMCs transfected with Ctr-siRNA or Ube2i-siRNA, with or without DHA (50 μM) treatment (n = 4). p value versus Ctr-siRNA by one-way ANOVA tests. C–E Western blotting and quantification of UBC9, IL-6, and MCP-1 expression in AMPKα2−/− VSMCs transfected with Ctr-siRNA or Ube2i-siRNA before or after PA (300 μM) and DHA (50 μM) treatment for 1 h (n = 2). F, G Western blotting and quantification of c-myc, UBC9, and SUMO2/3 expression in AMPKα2−/− VSMCs transfected with Ctr-siRNA or Myc-siRNA (n = 4). H–K Western blotting (H) and quantification of tGFP (I), UBC9 (J), and SUMO2/3 (K) expression in 293T cells transfected with WT C-myc (WT), S64A/C-myc (S64A), or S67A/C-myc (S67A) vectors and treated for 2 hrs with AICAR (1 mM) (n = 2). L Extracts from 293T cells transfected with either WT, S64A, or S67A c-myc constructs were prepared to assess the interactions between the c-myc-tGFP fusion protein and AMPKα2 or pAMPKα. M Recombinant AMPK and recombinanted c-myc-tGFP protein were incubated in the kinase buffer with ATP. Immunoprecipitation was done using anti-tGFP antibody. N The phosphorylation of serine at amino acids 67 in the peptide PTPPLSPSRRSG by LC-MS/MS followed processed using Proteome Discoverer 1.3. O Representative images of immunostaining showing AMPKα2 and UBC9 expression and localization in human artery tissues from control (n = 4) and CAD patients (n = 8). Scale bar, 400 μM. P Semi-quantification analysis of immunostaining using Image-Pro plus software. Correlational analyses of AMPKα2 expression and the expression of UBC9 (Q) in human artery tissues. All data shown represent one of three separate experiments. Quantification of western blots was performed using Image-Pro plus software, distinctly from loading controls. Data are presented as the mean ± s.e.m. p value by two-sided Student’s t tests. AICAR 5-aminoimidazole-4-carboxamide 1-α-d-ribofuranoside, Ube2i-siRNA siRNA against mouse Ube2i, Myc-siRNA siRNA against mouse Myc, CAD coronary artery disease.

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