Rabbit α g6pdh

For whole cell extracts, cells were grown to exponential phase in SCD. 0.25 OD600 cells were pelleted, washed with dH20, and extracts were prepared by alkaline extraction (0.255M NaOH, 1% 2-mercaptoethanol) followed by precipitation in 9% trichloroacetic acid. Precipitates were washed with acetone, dried, and resuspended in 50μL Laemmli protein sample buffer prior to Western analysis.
Whole cell lysates or crude mitochondria extracts were freshly prepared and incubated at 70°C (to enable optimal detection of Mdi1*-2xFLAG) or 95°C for 10 minutes prior to SDS-PAGE, transferred to 0.2μm pore size PVDF membranes, and immunoblotted with the following primary antibodies: mouse α-FLAG (1:1000, Sigma F1804), rabbit α-G6PDH (1:2000, Sigma A9521), mouse α-Porin (1:2000; ThermoFisher 459500), rabbit α-Fzo1 (1:1000, a kind gift of Jodi Nunnari, Altos Labs), α-Aco1 (1:10000, a kind gift of Anju Sreelatha, UT Southwestern), α-Mia40 (1:10000, a kind gift of Anju Sreelatha, UT Southwestern). To detect Mdi1*-2xFLAG, goat anti-rabbit HRP (Sigma) was used (1:10000) and signal was visualized with SuperSignal West Femto Substrate (ThermoFisher). All other proteins were detected with secondary antibodies conjugated to DyLight800 (1:10000, ThermoFisher). Images were acquired with a ChemiDoc MP Imaging System (BioRad). Linear adjustments to images were made with Photoshop (Adobe).