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Mitochondrial Complexes Profiling in Macrophages

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Protein expression in stimulated macrophages was determined by western blot using 20 μg of total protein per sample^{3 (link)}. Separation into cytoplasmic and nuclear fractions was performed using 0.4 M NaCl and 0.1% Nonidet P-40 (NP-40) in extraction buffer^{57 (link)}. SDS immunoblots (IBs) were performed to evaluate steady-state protein levels of structural subunits of mitochondrial complexes assessed by native gels. Antibodies in this study are as follows: anti ACO1/2, anti IDH1, anti SDHB, anti SDHA, anti NDUFS1, anti MTCO1, anti ATP5A, anti NDUFS8, anti total OXPHOS (Complex V, ATP5A subunit; Complex IV, COXII subunit; Complex III, UQCRC2 subunit; Complex II, SDHB; Complex I, NDUFB9 subunit) and anti Phospho-S²⁹³ PDHE1-alpha were from Abcam; anti-FECH antibody was from Proteintech; anti phospho-, total PDK1 and anti HIF1α were from Novus Biologicals. For loading control anti CS, anti TOM20, Lamin B and anti β-actin were used (respectively from Sigma, Santa Cruz, Abcam, and Millipore).

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Palmieri E.M., Gonzalez-Cotto M., Baseler W.A., Davies L.C., Ghesquière B., Maio N., Rice C.M., Rouault T.A., Cassel T., Higashi R.M., Lane A.N., Fan T.W., Wink D.A, & McVicar D.W. (2020). Nitric oxide orchestrates metabolic rewiring in M1 macrophages by targeting aconitase 2 and pyruvate dehydrogenase. Nature Communications, 11, 698.

Publication 2020

anti HIF1α anti TOM20 Lamin B anti β-actin

Actin Anti antibody Antibodies Biologicals Complex i Complex ii Complex iii Complex iv Complex v Coxii Cytoplasmic Gels Hif1α Immunoblots Lamin b Macrophages Mitochondrial Nacl Nonidet p 40 Novus Pdk1 Protein Sdhb Subunit Western blot

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Variable analysis

independent variables

Stimulation conditions of macrophages

dependent variables

Protein expression levels of structural subunits of mitochondrial complexes
Phosphorylation level of PDHE1-alpha (at Ser293)

control variables

Total protein amount per sample (20 μg)
Cytoplasmic and nuclear fractionation procedure (0.4 M NaCl and 0.1% Nonidet P-40 in extraction buffer)

controls

Positive controls: Anti-ACO1/2, anti-IDH1, anti-SDHB, anti-SDHA, anti-NDUFS1, anti-MTCO1, anti-ATP5A, anti-NDUFS8, anti-total OXPHOS antibodies
Negative controls: Anti-CS, anti-TOM20, anti-Lamin B, anti-β-actin antibodies

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