mtatp8  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Calorie Restriction Induces Degeneration of Neurons with Mitochondrial DNA Depletion by Altering ER-Mitochondria Calcium Transfer

doi: 10.1101/2024.06.14.599123

Figure Lengend Snippet: CR induces degeneration of UL12.5 expressed rho - neurons. A) Representative images of a fixed UL12.5-DsRed (red) expressing neuron with mitochondrial marker protein TOM20 (green). Scale bar: 10 μm. B) Quantification of the ratio of mtDNA area to mitochondrial area. Error bars: SD (n = 10 neurons; ***P < 0.001). C) qPCR was used to detect the relative mtDNA copy number ( MTND1 , MTND4 ) in control or UL12.5-DsRed expressing neurons. Error bars: SEM (n = 3; ***p < 0.001). D) Western blot analysis of mtDNA-encoded protein (MTATP8) and nuclear DNA-encoded mitochondrial protein (ATP5A). E, F) Mitochondrial respiration was abrogated in UL12.5 expressing neurons. Neurons were infected with mito-DsRed or UL12.5-DsRed before OCR measurement. Oligomycin (inhibiting ATP synthesis), FCCP (an uncoupler inducing maximum respiration), and antimycin A & rotenone (inhibiting total mitochondrial respiration) were added sequentially (E). (F) Proton leak (subtraction of the antimycin A & rotenone OCR from the oligomycin OCR) and mitochondrial ATP production capacity (subtraction of the oligomycin OCR from the basal OCR). Error bars: SEM (n ≥ 3; ***P < 0.001). G, H) Control or rho - neurons were treated with 2-DG for 0, 2, and 4 days. Representative brightfield images are shown in (G). Scale bar: 25 μm. Quantification of the fraction of neurites with beads (H). Error bars: SD (n = 4; ***p < 0.001). I) Quantification of the MTT reduction in control and rho - neurons. Error bars: SD (n = 3; **p < 0.01). J, K) Representative immunofluorescence images of synapses (SYN1, green) and nuclei (DAPI, blue) (J), scale bar: 10 μm. Quantification of the ratio of SYN1 spots to cells (K). Error bars: SD (n = 3; ***p < 0.001). L, M) Representative brightfield images of control and rho - neurons treated with high-glucose or low glucose for 4 days (L). Scale bar: 25 μm. Quantification of the fraction of neurites with beads (M). Error bars: SD (n = 3; ***p < 0.001). N, O) Representative brightfield images of control and rho - neurons treated with rapamycin for 4 days (N). Scale bar: 25 μm. Quantification of the fraction of neurites with beads (O). Error bars: SD (n = 3; ***p < 0.001).

Article Snippet: The primary antibodies were Rabbit polyclonal anti ATP5A (Proteintech, 14676-1-AP, 1:1000), Rabbit polyclonal anti MTATP8 (Santa Cruz, sc-84231, 1:1000), Rabbit polyclonal to Tubulin β-3 (TUBB3) (Biolegend, 802001, 1:1000) and Rabbit polyclonal to GAPDH (Abcam, ab9485, 1:1000), Rabbit polyclonal anti MTCOX3 (Proteintech, 55082-1-AP,1:500), Mouse monoclonal anti FLAG (Sigma-Aldrich, F1804, 1:1000).

Techniques: Expressing, Marker, Control, Western Blot, Infection, Immunofluorescence

Journal: Metabolites

Article Title: Differential Inhibition of Anaplerotic Pyruvate Carboxylation and Glutaminolysis-Fueled Anabolism Underlies Distinct Toxicity of Selenium Agents in Human Lung Cancer

doi: 10.3390/metabo13070774

Figure Lengend Snippet: Protein Targets and antibodies used for RPPA analyses.

Article Snippet: MTATP8 , Proteintech Group , 26723-1-AP , 1:100.

Techniques:

Journal: Scientific Reports

Article Title: NADPH oxidase is implicated in the pathogenesis of oxidative phosphorylation dysfunction in mice fed a high-fat diet

doi: 10.1038/srep23664

Figure Lengend Snippet: ( a ) BN-PAGE analysis of OXPHOS complexes in WT and NADPHox-deficient (NOX −/− ) mice fed a standard chow (S) or a high-fat (F) diet. A Western blot analysis of mitochondrial proteins was performed using antibody against complex I subunit NDUFA9, complex II subunit SDHA, complex III subunit UQCR2 protein, complex IV subunit MTCO1, complex V subunit ATP5A1, and TOM complex subunit TOM20. The expression of TOM complex (TOM) was used as loading control. ( b ) The mitochondrial proteins extracted from the same groups of mice were separated in the first dimension using BN-PAGE, and then in the second dimension using SDS-PAGE. The presence of individual subunits of these complexes was identified by immunoblotting using appropriated antibodies. The expression of VDAC1 was used as loading control. X-fold, amount of subunit in HFD-fed mice (WT or NOX −/− ) divided by amount of same subunit in control mice, whether WT/SCD or NOX −/− /SCD.

Article Snippet: Western blotting was performed using primary antibodies against subunits NDUFA9, NDUFA6, NDUFB6, NDUFS3, NDUFV1, NDUFV2, MTND1, MTND4L, and MTND6 (complex I); SDHA (complex II); UQCRC2, UQCRFS1, and MTCYTB (complex III); COX4 and MTCO1 (complex IV); ATP5A1 and MTATP8 (ATP synthase) (Molecular Probes Inc. Eugene.

Techniques: Western Blot, Expressing, SDS Page