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.