Fig. 6: dGTP binds to the dNK domain of NDUFA10 subunit.

a Analysis of NDUFA10 protein levels in whole cell lysates (by western blot; left panel) and in mitochondria (by BN-PAGE; right panel) of wild type (NDUFA10^WT), NDUFA10 knock-out (NDUFA10^KO), and NDUFA10 knock-in (NDUFA10^KI) HEK-293T cells, and NDUFA10^FLAG HeLa cells. Immunodetection with anti-NDUFA10 and anti-GAPDH antibodies. b Ratio between complex I enzyme activity and citrate synthase activity in NDUFA10^WT, NDUFA10^KI, and NDUFA10^KO cells. Results are mean values of 5 independent experiments (circles). Error bars represent standard deviations. c Autoradiography image of photoaffinity labeling with immunopurified NDUFA10^KI and growing concentrations of [α-³²P]-dGTP (20 nM, 200 nM, and 2 µM). NDUFA10^FLAG was incubated with 20 nM [α-³²P]-dGTP. d Western blot analysis of NDUFA10 pulled-down with ɣ-amino-octyl-dGTP on native mitochondrial extracts from NDUFA10^WT and NDUFA10^FLAG cells. Elution was performed using growing concentrations of dGTP (0.1, 1, and 10 mM) and immunodetection with anti-NDUFA10 antibody. e Mitochondrial dNTP levels in NDUFA10^WT, NDUFA10^KO, and NDUFA10^KI cells. Total dNTPs in TCA extracts (pmol dNTP/mg of protein) (upper panel) and ratio of protein bound to free dNTP fractions (bottom panel). N = 3–8 independent experiments. Two-tailed Mann-Whitney U test, *p < 0.05, **p < 0.01, ***p < 0.005.