Complex 1 enzyme activity kit

Mitochondria were isolated from Wt and Akap1^−/− lungs using the mitochondria isolation kit as per the manufacturer’s instructions (Thermo Scientific). Mitochondrial pellets were lysed in 2% CHAPS in TBS supplemented with protease and phosphatase inhibitors, quantified by Pierce BCA protein assay kit (Thermo Scientific) and stored at −80°C. The fumarase activity colorimetric assay kit MAK206 (Sigma-Aldrich, St. Louis, MO), citrate synthase activity kit MAK193 (Sigma-Aldrich) and aconitase activity assay kit MAK051 (Sigma-Aldrich) were used to detect enzymatic activity in Wt and Akap1^−/− mitochondrial fraction (25 μg) according to the manufacturer’s instructions. Aconitase activity (nmole/min/ml), citrate synthase activity (milliunit/mL) and fumarase activity (milliunit/mL) was normalized to the amount of protein used in the assay. Mitochondrial Complex I activity was determined using 25 μg of mitochondrial lysate and Complex I enzyme activity kit (#ab109721, Abcam, Waltham, MA) as per manufacturer’s instructions. The Complex I activity (mOD/min) was normalized to the amount of protein used in the assay.

Mitochondrial complex I activity was measured using the Complex I Enzyme Activity Kit (#ab109721, Abcam, UK) following the manufacturer's directions. PBLs were treated with 1 mM paraquat for 24 h, harvested by centrifugation at 1000 g, and washed twice with ice-cold PBS. Mitochondria were isolated using the Mitochondria Isolation Kit (Abcam). Purified mitochondria were suspended in 500 μL Buffer C and lysed for 30 min by adding the detergent provided in the kit. After centrifugation, the mitochondrial proteins were quantified using the Bradford assay, and mitochondrial lysates were adjusted to the same protein concentration (approximately 1 mg/ml). Serial dilutions were prepared in the incubation solution (Abcam CI Assay Kit). Complex I activity was measured using 200 μL aliquots of the serial dilutions, each in triplicate, using the Molecular Devices VERSAmax microplate reader at 450 nm, with kinetic readings taken every 30 sec for 30 minutes. Activity was represented as mOD/min and normalized to the control group (defined as 1.00).

The RPE/retina was isolated and protein extracted in 100 μl T-PER buffer (Pierce Biotechnology). Tissues were homogenized with a glass tissue grind tube (Kontes) on ice. The supernatant was collected after centrifugation at 13,500 g for 10 min. The protein concentration was detected by BCA assay (Thermo Fisher Scientific) and 110 μg protein from each sample was used for the experiment. The activity of complex I was measured using a Complex I Enzyme Activity Kit (Abcam) according to the manufacturer's protocol. Absorbance at 450 nm was measured in a transparent flat-well plate at 30 s intervals for 30 min. The activity of complex I was presented as rate/slope (mOD/min) per 110 μg of protein.