Complex 1

For the respiratory chain supercomplex analysis, mitochondria were resuspended in 100 μl of a buffer composed of 1.5 M aminocaproic acid, 50 mM Bis-Tris, pH 7.0, solubilized with 1% (w/v) digitonin, incubated on ice for 5 min and solubilized protein complexes were separated through centrifugation at 20,000 × g for 30 min at 4 °C.
Supernatants were collected, supplemented with a sample buffer prepared with 5% (w/v) Serva Blue G, 1.0 M aminocaproic acid and quickly loaded onto a 3–12% blue native polyacrylamide gradient gel (BN-PAGE, Invitrogen). After electrophoresis, gels were used for Western Blotting against Ndufs1 (Complex I, Abcam), OSCP, IF1 or SDHA (Complex II, Santa Cruz Biotechnology) subunits.

Mitochondrial OXPHOS Complex I enzyme activity was measured by a Complex I (Abcam, cat: ab109721) Enzyme Activity Microplate Assay Kit according to the manufacturers' protocols. Briefly, isolated mitochondria samples were prepared using mitochondrial lysis kit (GENMED, cat: GMS10018V.A) at final protein appropriate concentration respectively, and the samples were loaded to the wells of the microplate. The microplate was incubated and then assay solution was added to each well. Optical density was measured in a kinetic mode at appropriate temperature for suitable minutes.

Activity of complex I and complex IV was assessed according to the protocol of the Complex I (ab109720)/Complex IV (ab109876) Enzyme Activity Dipstick Assay Kit (Abcam, Cambridge, United Kingdom). In this technique the proteins from cellular lysates migrate through a nitrocellulose membrane. Then, Complex I is immunocaptured (i.e., immuno-precipitated in active form) on the dipstick. Then, the dipstick is immersed in Complex I activity buffer solution containing NADH as a substrate and nitrotetrazolium blue (NBT) as the electron acceptor. Immunocaptured complex I oxidizes NADH and the resulting H+ reduces NBT to form a blue-purple precipitate at the Complex I antibody line on the dipstick. The signal intensity of this precipitate corresponds to the level of Complex I enzyme activity in the sample. Dipsticks images were taken with a Molecular Imager ChemiDoc XRS+ System (BIORAD, Hercules, CA, United States) and quantified by the Image Lab software.