Mice were euthanized post-IR injury, and the AAR was quickly removed, frozen, and pulverized. Pulverized tissue was then treated according to manufacturers’ protocol (Abcam), with minor modifications. To prevent in vitro posttranslational modifications during sample preparation, apyrase (1×) and phosphatase inhibitor cocktails 2 and 3 (Millipore Sigma) were added to the solubilizing buffer. Apyrase is an enzyme that catabolizes the hydrolysis of ATP and therefore depletes ATP availability for de novo phosphorylation events (42). A PDH enzyme activity microplate assay kit (Abcam) was used to measure enzyme activity. To stay within the linear range of the assay for mouse cardiac tissue, we loaded 60 μg of lysate per well (as shown in the manufacturer’s manual). Recordings were acquired on a Tecan Infinite M1000Pro plate reader at 450 nm every minute for 15 min with a 3-s mix between measurements.

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