Phospho-FUNDC1 (Ser17) antibody - #AF0001

Fig. 9: Insights into the role of CD36 palmitoylation in cardiomyocyte autophagy through PGAM5. a Western blotting analysis of LC3II and SQSTM1 protein levels in myocardium from WT-CD36 and AA-SS-CD36 mice. n = 7. b Western blotting analysis of LC3II and SQSTM1 protein levels in cardiomyocytes transfected with WT-CD36 or AA-SS-CD36 for 48 h. n = 10. c Overlap between CD36 binding proteins, as assessed by CoIP-MS analysis and proteins in the Autophagy Database (http://autophagy.info/). d Endogenous CD36 immunoprecipitation followed by anti-CD36 and anti-PGAM5 western blot analysis of cardiomyocytes (NMVCs). n = 4. e Quantification of Cyto-ID staining in cardiomyocytes (NMVCs) transfected with WT-CD36 or AA-SS-CD36 for 48 h, and then treated with 500 nM rapamycin (Rapa) and 60 µM CQ, the combination for 16 h. n = 6 per group. f–h Western blotting analysis of the mitochondria expression of CD36 and PGAM5 in cardiomyocytes (NMVCs) transfected with WT-CD36 or AA-SS-CD36. n = 7 per group. i Relative PGAM5 mRNA levels in cardiomyocytes transfected with WT-CD36 or AA-SS-CD36 for 48 h. n = 5. j Time course of PGAM5 protein stability in cardiomyocytes (NMVCs) transfected with WT-CD36 or AA-SS-CD36 after treatment with CHX (10 µg/ml). n = 6. k Ubiquitination of exogenous PGAM5 in cardiomyocytes (NMVCs) transfected with WT-CD36 or AA-SS-CD36. Data are representative of six independent experiments. l The mitochondria expression of phosphorylation Fundc1 and total Fundc1 protein in cardiomyocytes (NMVCs) transfected with WT-CD36 or AA-SS-CD36. n = 6 for phosphorylation Fundc1; n = 7 for total Fundc1. Data are presented as means ± SEM. Statistical significance was assessed by Kruskal-Wallis, followed by false discovery rate [FDR] method of Benjamini and Hochberg test (a), two-tailed unpaired Student t test (b, d, g, i and l), two-way ANOVA, followed by Tukey post hoc multicomparisons test (e, j) and two-tailed Mann-Whitney U test (h). Source data are provided as a Source Data file.

Figure 3. Construction of In Vitro Model and Observation of Mitochondrial Autophagy and Expression Changes of c-FLIP, JNK, and FUNDC1 (A and B) Optical microscopy of cardiac microvascular endothelial cell (CMEC) morphology and CCK-8 assay for cell proliferation after 12 hours of different lipopolysaccharide (LPS) concentrations. (C and D) Optical microscopy of CMECs morphology and CCK-8 assay for cell proliferation at various times after 10 μg/mL LPS treatment. (E and F) c-FLIP expression at various times after 10 μg/mL LPS treatment. (G) Transmission electron microscopy (×12,000) of CMECs microstructure to assess mitochondrial damage and autophagy (representative images). (H to L) Western blot of mitochondrial autophagy-related proteins. (M-Q) Expression of c-FLIP, total c-Jun N-terminal kinase (t-JNK), phosphorylated JNK (p-JNK), total Fun14 domain-containing protein 1 (t-FUNDC1), phosphorylated FUNDC1 (p-FUNDC1) proteins, and FUNDC1 mRNA. Note: n = 3/group; 3 technical replicates; mean ± SEM; Student's t-test (N-Q), analysis of variance with Tukey’s post hoc test (B, D, F, and I to L). ∗P < 0.05 vs Ctrl (control) group, ∗∗P < 0.01 vs Ctrl group, ∗∗∗P < 0.001 vs Ctrl group. Abbreviations as in Figure 1.

Fig. 6 NTRK1 silencing might suppress mitophagy in mouse neurons through the inactivation of the AMPK/ULK1/FUNDC1 pathway. A, B WB revealed that O304 treatment abrogated the suppression effect of NTRK1 knockdown on the activity of the AMPK/ULK1/FUNDC1 pathway in mouse neurons. n = 3. C, D WB also indicated that O304 treatment counteracted the suppression effect of NTRK1 silencing on mitophagy in mouse neurons. n = 3. E, F The results of TOMM20/LC3-II fluorescence staining revealed that the O304 treatment reversed the suppression effect of NTRK1 knockdown on the expression of Parkin in mouse neuronmitochondria. n = 3.

Figure 1 Expressions of inflammatory cytokines, HIF-1α and FUNDC1 in healthy and pulpitis tissues. (A) mRNA expression of IL-1β, IL-6, IL-8 and TNF-α in human healthy and pulpitis tissues. mRNA expression of (B) HIF-1α and (C) FUNDC1 in human healthy and pulpitis tissues. (D) Representative immunostaining images of HIF-1α and FUNDC1 in human healthy or inflamed dental pulp tissues. Scale bars are 100 and 25 µm, respectively. Results are presented as the means ± SD from ≥ three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. healthy. HIF-1α, hypoxia-inducible factor-1α; FUNDC1, FUN14 domain-containing 1.