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The first injection in port A contained the substrate for each individual respiratory chain complex (I: 5 mM pyruvate + 2.5 mM malate, II: 10 mM succinate + 1 M rotenone, III: 0.5 mM duroquinol, IV: 0.5 mM TMPD + 2 mM ascorbic acid) along with 1 mM ADP and 10 nM recombinant perfringolysin O (Agilent Technologies)