Yeast Cell Extract Preparation and Immunoblotting

Cell extracts for expression analysis were prepared as described (14 (link)). Briefly, yeast were grown in selective SD media to mid-exponential phase, whereafter 2.5 A₆₀₀ units were harvested and lysed by an alkaline lysis method (45 (link)). Lysates were resuspended in SDS sample buffer and incubated at 37 °C for 30 min, centrifuged to remove cell debris, and analyzed by SDS-PAGE and immunoblotting.
Immunoblotting was performed as described (46 (link)) with the following antibodies: rabbit anti-Doa10 at 1:2000 (27 (link)); mouse anti-MYC (9E10, Covance) at 1:10,000; rabbit anti-G6PDH (A9521, Sigma) at 1:10,000; mouse anti-FLAG (F3165; Sigma) at 1:2000 or 1:10,000; peroxidase anti-peroxidase (Sigma) at 1:1000; mouse anti-PGK (459250, Thermo Fisher Scientific) at 1:20,000; rabbit anti-Dfm1 at 1:2000 (47 (link)); rabbit anti-Ubc6 at 1:2000 (31 (link)). Primary antibody incubations were followed by peroxidase-coupled sheep anti-mouse or peroxidase-coupled goat anti-rabbit secondary antibodies (GE Healthcare) at 1:10,000 and visualized by enhanced chemiluminescence (48 (link)) on a G:Box system (Syngene).