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. 2022 Mar 1;8:21. doi: 10.1038/s41421-021-00363-1

Fig. 6. Naringenin directly bound to 14-3-3ε and inhibited its interaction with TFEB.

Fig. 6

a Workflow of the naringenin-interactome approach. b Isothermal titration plot of naringenin (in cells) with 14-3-3 (in syringes). The inset shows a graphic representation of the different thermodynamic parameters analyzed. The solid line represents the best nonlinear least-squares fit to a single binding site model. c, d CETSAs of 14-3-3ε in the absence and presence of naringenin at different temperatures (c) and different doses (d), the results were evaluated by western blots. The experiments were performed in macrophages on three independent occasions. e Co-IP analysis of 14-3-3ε and TFEB in the presence or absence of naringenin in 293A cells transfected with Flag-TFEB plasmid and His-14-3-3ε plasmid. Upper, the lysates were immunoprecipitated with anti-His antibody, and the precipitates were analyzed by immunoblotting with anti-Flag antibody. Lower, the lysates were immunoprecipitated with anti-Flag antibody, and the precipitates were analyzed by immunoblotting with anti-His antibody. f Results of the competitive binding assay for naringenin of the p-TFEB peptide (LVGVTSSpSCPADLTQ) and 14-3-3ε. The probe concentration was 10 nM, and the concentration of 14-3-3ε was 10 nM. Data are presented as the means ± SEM of three independent experiments.