Protein expression levels measurement by Western blotting
To check for protein expression levels, yeast cells were transformed with the corresponding plasmids and grown as specified in a previous section. Two optical density (OD) units were harvested by centrifugation. Total proteins were extracted from the equivalent number of cells and loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be separated according to their molecular weight. Next samples were immunoblotted on a nitrocellulose membrane for 90 min at 100 V on ice, as previously described (Ishikawa et al., 1997). Antibodies against FLAG (Sigma-Aldrich), PGK (Invitrogen) and Xrn1 (a gift from A. Johnson) were used. Detection of proteins was performed with an Amersham Imager 600.