ATP5A1 Antibody - #DF3806

Figure 6 The TCN2 effect allele exacerbates mitochondrial defect in renal tubular cells under high-glucose conditions (A and B) Western blot analysis of subunits of mitochondrial oxidative phosphorylation (OXPHOS) complexes, outer mitochondrial membrane, and inner mitochondrial membrane protein in primary tubular cells from TCN2 K77M mice and WT mice with or without high glucose, n = 3 in each group. Data are represented as mean ± SEM. (C) Statistical analysis of the differences in basal OCR, maximal OCR, respiratory reserve capacity, and nonmitochondrial OCR of cultured primary tubular cells from TCN2 K77M mice and WT mice with or without high glucose, n = 3 in each group. Data are represented as mean ± SEM. (D) Immunofluorescence staining of MitoTracker in cultured primary tubular cells from TCN2 K77M mice and WT mice with or without high glucose. Scale bars: 100 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, according to two paired samples with a Wilcoxon test. Discussion

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 4 CHCHD2 promotes F1F0-ATPase assembly in SH-SY5Y cells treated with MPP+.Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells; CHCHD2-Flag: LV-CHCHD2-Flag–transfected SH-SY5Y cells; CHCHD2-T61I: LV-CHCHD2-T61I-Flag-transfected SH-SY5Y cells; untreated: cells cultured in MEM-F12 medium; MPP+-treated: cells cultured with MPP+ (500 μM for 24 hours). (A) Representative western blots of ATP5A1, ATP6, VDAC1, and α-tubulin in the cytoplasm and mitochondrial membrane fractions without MPP+ treatment. (B) The ratio of ATP5A1 in the mitochondrial membrane and cytoplasmic fractions of control cells. (C) The ratio of ATP6 in the mitochondrial membrane and cytoplasmic fractions of control cells. (D) Representative western blots of ATP5A1, ATP6, and β-actin in whole-cell lysates from cells without MPP+ treatment. (E) Quantification of ATP5A1 and ATP6 expression in whole-cell lysates of control cells. (F) Representative western blots of ATP5A1, ATP6, VDAC1, and α-tubulin in the cytoplasmic and mitochondrial membrane fractions of cells treated with MPP+. (G) The ratio of ATP5A1 in the mitochondrial membrane and cytoplasmic fractions of MPP+-treated cells. (H) The ratio of ATP6 in the mitochondrial membrane and cytoplasmic fractions of MPP+-treated cells. (I) Representative western blots of ATP5A1, ATP6, and β-actin in whole-cell lysates of cells treated with MPP+. (J) The relative expression levels of ATP5A1 and ATP6 in whole-cell lysates of MPP+-treated cells. Data are expressed as the mean ± SD (n = 3 independent experiments). **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). Mito-mem: Mitochondrial membrane; MPP+: 1-methyl-4-phenylpyridinium; VDAC1: voltage-dependent anion-selective channel protein 1.