Fig 2: PKA addition to mitochondrial import experiments increased the activity of complex I and complex I/III. RRL translation mixtures containing newly synthesized [35S]-Met labeled NDUFS4 protein or [35S]-Met-labeled NDUB11 protein or no protein present (Blank) were added to the import mixture containing RLM in absence and in the presence of PKA. After 60 min incubation at 30 °C, mitochondria were spun down from the import mixture and used for spectrophotometric analysis. (A) Activity of complex I (NADH-UQ oxidoreductase). (B) Activity of complex III (ubiquinol-cytochrome c oxidoreductase). (C) Activity of complex I/III (NADH-cytochrome c oxidoreductase). The values are the means ± SD of three different experiments. (NDUFS4 + PKA vs. NDUFS4, * p < 0.05, ** p < 0.01, Student’s t-test). For more details, see under Materials and Methods.

Fig 3: PKA addition to mitochondrial import experiments increased supercomplexes containing complex I and complex III. RRL translation mixture containing the newly synthesized [35S]-Met labeled NDUFS4 protein was added to the import mixture containing RLM in absence (CTRL) and in the presence of PKA. After 60 min incubation at 30 °C, mitochondria were spun down. The solubilized pellets were analyzed by 1D-BNE/2D-SDS PAGE. (A) Representative images of autoradiography. The histograms represent the percentage of ADU of autoradiography revealed spots ([35S]-Met NDUFS4) in free complex I (CxI) and in supercomplexes (SCs) of PKA-treated samples with respect to untreated cells (CTRL). The complex alignment of radioactive spots was performed by using first dimension radioactive bands of the same lanes. (B) Representative images of immunoblotting analysis with antibodies specified in the figure. The histograms represent the percentage of ADU of PKA-treated samples with respect to untreated cells (CTRL) of immuno-revealed spots by antibodies against NDUFA9 and CORE II in free complex I (CxI) and complex III (CxIII) and in supercomplexes (SCs). The complex alignment of immuno-revealed spots was performed by using the ß-subunit antibody revealing the dimeric form of ATP synthase (DCxV). The values are the means ± SD of three different experiments. (PKA vs. CTRL, * p < 0.05, ** p < 0.01, Student’s t-test). For more details, see under Materials and Methods.

Fig 4: Isoproterenol increased supercomplexes containing complex I, complex III and complex IV. The H9c2 cell cultures were incubated for 30 min in absence (CTRL) or in the presence of isoproterenol (ISO). Mitochondrial solubilized proteins were separated by 1D-BNE/2D-SDS-PAGE and transferred into nitrocellulose membrane, followed by immunoblotting analysis with specific antibodies. (A) Representative images of immunoblotting with antibody against NDUFS4. (B) Representative images of immunoblotting with antibodies against Core II and ß-ATP synthase. (C) Representative images of immunoblotting with antibody against Cox IV. (A–C) the histograms represent the percentage of ADU of isoproterenol-treated cells (ISO) with respect to untreated cells (CTRL) of immuno-revealed spots in free complexes (CxI, CxIII, CxIV) and in supercomplexes (SCs). The values are the means ± SD of three different experiments. (ISO vs. CTRL, *** p < 0.001, Student’s t-test). For more details, see under Materials and Methods.

Fig 5: PKA addition to mitochondrial import experiments increased the complex I-supporting respiration. RRL translation mixtures containing newly synthesized [35S]-Met-labeled NDUFS4 protein or no protein present (Blank) were added to the import mixture containing RLM in absence and in the presence of PKA. After 60 min incubation at 30 °C, mitochondria were spun down from the import mixture and used to measure the oxygen consumption rates. (A) Oxygen consumption rates using pyruvate/malate (P/M) as substrate. (B) Oxygen consumption rates using succinate as substrate. The values are the means ± SD of three different experiments. (NDUFS4 + PKA vs. NDUFS4, * p < 0.05, Student’s t-test). For more details, see under Materials and Methods.