SUPPLEMENTARY METHODS
Activities of mitochondrial ETC complexes I, II, III, IV and V
Complex I activity: Capture antibody for complex I was pre-coated in the wells of the microplate, and the samples containing 50 µg proteins were added to the wells. In this assay, complex I activity is measured by the oxidation of NADH to NAD
+
, which leads to increased absorbance at 450 nm.
Complex II activity: Capture antibody for complex II was pre-coated in the wells of the microplate, and the samples containing 25 µg proteins were used for this assay. In this assay, the production of ubiquinol by complex II protein is related to the reduction of the 2,6diclorophenolindophenol (DCPIP, blue) to DCPIPH
2
(colorless), and the absorbance is measured at 600 nm.
Complex III activity: The mitochondria were directly used for this assay. Complex
III activity was measured by monitoring the conversion of cytochrome c from its oxidized form to its reduced form, as a linear increase in absorbance at 550 nm. In this assay, rotenone was added to inhibit complex I so that all of the reduction of cytochrome c was via complex
II. In addition, KCN was added to inhibit complex IV so that there was no re-oxidation of cytochrome c by this enzyme.
Complex IV activity: Capture antibody for complex IV was pre-coated in the wells of the microplate, and 5 µg proteins from the samples were used for this assay. The activity of complex IV was determined colorimetrically by following the oxidation of reduced cytochrome c as a decrease in absorbance at 550 nm.
Complex V activity: ATP synthase is immunocaptured within the wells of the microplate, which has a monoclonal antibody pre-bound to the wells. 50 µg proteins from the
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samples were used for this assay. The ATP synthase complex is responsible for ATP production in the oxidative phosphorylation process, and it can work in reverse as a proton pumping ATPase. In this assay, the conversion of ATP to ADP is coupled to the oxidation of
NADH to NAD + , which is monitored as a decrease in absorbance at 340 nm.
Activity of mitochondrial citrate synthase
Citrate synthase was solubilized by adding extraction buffer (9 volumes) to the mitochondria in the samples, and the samples containing 10 µg proteins were used for this assay. The activity of citrate synthase was determined in an immunocapture based manner by recording the color development of 5-thio-2-nitrobenzoic acid (TNB) at 412 nm, which was generated from 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) present in the reaction of citrate synthesis.
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SUPPLEMENTARY FIGURE
Fig. S1. Activities of ETC complexes and PDH in the frontal cortex of the children (a) and adults (b) with autism and their age-matched control subjects.
Activities of complexes I, V, and PDH were significantly decreased in the children with autism compared to the control group (a). *, p <0.05: **, p <0.01
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