FUNDC1 Antibody - #AF0002

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.

Fig. 5: Cross-species MSR signature attenuates mitochondrial dysfunction. a, c Effects of NMN on the expression level of proteins involved in UPRmt and mitophagy in the 5xFAD mice hippocampi of individuals with and without NMN (n = 3 biologically independent samples; two-sided unpaired t-test). b, d Corresponding quantification of western blot data of (a, c) (n = 3 mice in each group). Data were analyzed by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test. e, f Representative immunostained images (e) and quantification (f) of ATF4 in the hippocampi of AD (VEH) and AD (NMN) tissues (n = 3 mice; two-sided unpaired t-test). Scale bar, 100 μm. g The mRNA expression measurement of MSR signature after supplementing with NMN in 5xFAD mice (n = 3; two-way ANOVA). h Synaptic proteins from the 6-month hippocampi were analyzed by Western blotting (n = 3 mice in each group). Data were analyzed by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test. i–k we obtained T2-weighted anatomical images to analyze the structure difference in the ventricle system using a 9.4 Tesla magnetic resonance imaging (MRI) scanner. Data were pooled from at least 3 biological replicates. Data were analyzed by two-sided one-way ANOVA followed by Tukey’s multiple comparisons test. l Mito-nuclear protein imbalance evaluated by the ratio of mitochondrial DNA (mtDNA)-encoded protein (MTCO1) and nuclear DNA (nDNA)-encoded protein (ATP5a) in three groups (n = 3 mice; one-way ANOVA). m, n Representative immunostaining and quantification of the MitoSOX in the cultured primary astrocytes (n = 6 mice; two-sided unpaired t-test). Scale bar, 20 μm. o, p Representative electron microscopic images, and multiple quantifications, showing the effects of NMN on mitochondrial morphology in mouse hippocampal brain tissues. The quantification was obtained in two independent experiments performed in duplicates in at least 20 ROIs. q ATP Assay was performed in 6-month-old AD mice brain (n = 5 mice, two-sided unpaired t-test). All experiments were performed independently with at least three biological replicates with similar results. Data are shown as mean ± s.e.m. The p-values are indicated on the graphs. ns not significant.

Figure 6 Effects of Mo and/or Cd on immunofluorescence of LC3, mitophagy-related mRNA and protein expression levels. (A) The mRNA levels of FUNDC1, LC3A, LC3B and PGAM5. (B) The western blot results of FUNDC1, p-FUNDC1 and LC3. (C) The quantification of FUNDC1, p-FUNDC1 (Ser17) and LC3II/LC3I protein levels. (D) Immunofluorescence co-location of COX IV and LC3 at day 50. In the images, the nucleus staining is shown in blue, COX IV staining is shown in green, LC3 staining is shown in red, and the signals of colocalization are shown in merged images. (E) Pearson coefficient of the colocalization of COX IV and LC3. Data are expressed as means ± SD (n = 6). “*” indicates significant difference compared to the corresponding control (*P < 0.05, **P < 0.01). “#” indicates statistically significant difference between corresponding groups (#P < 0.05, ##P < 0.01).