Yeast Protein Synthesis Turnover Assay

Yeast expressing the indicated reporter substrate under the GAL1 promoter was grown in galactose-containing medium until mid-log phase. Protein synthesis was terminated by adding glucose (final concentration 2%) and cycloheximide (final concentration 0.1 mg/ml). One OD₆₀₀ unit was harvested at the indicated time points and total protein extracts were prepared by lysis and precipitation in 12.5% trichloroacetic acid. Total yeast lysates were separated by SDS-PAGE (NuPAGE, Invitrogen) followed by transfer to nitrocellulose membranes (PROTRAN; Schleicher & Schuell) or PVDF membrane (Millipore). Western blot analysis was performed as described previously56 (link) using the following primary antibodies: a mixed monoclonal antibody specific to GFP (Roche) (1:2000), a polyclonal anti-GFP antibody (Ab290, Abcam) (1:5000), monoclonal antibodies against β-actin (Abcam) (1:5000) or against PGK1 (Life technologies) (1:5000).