Ab113685

Newly synthesized yeast proteins were radioactively labeled in-vivo by a 7–10 minute pulse with [³⁵S] methionine in either 30°C or 37°C. Labeling was stopped by adding to the cells ice-cold TCA to a final concentration of 10%. Cells were then lysed and proteins were immunoprecipitated as previously described³⁶ with an antibody against RFP (Abcam, ab62341), HA (BioLegend, 901502), Kar2 (kindly provided by Peter Walter) or CPY (Abcam, ab113685). Protease inhibitors (Complete EDTA-free cocktail; Roche) were used throughout the extraction and immunoprecipitation process. Immunoprecipitated samples were resolved on polyacrylamide gels, which were then exposed to Phosphor Screen (GE Life Sciences) and scanned by phosphorimager. Translocation efficiency was calculated as
$\left[{\left(\frac{ER\mathit{\text{form}}}{\mathit{\text{total protein}}}\right)}_{\mathit{\text{mutant}}}/{\left(\frac{ER\mathit{\text{form}}}{\mathit{\text{total protein}}}\right)}_{WT}\right]$ . Differences were measures for statistical significance using two-tailed student t-test with unequal variance, as indicated in the figure legends. For the Tetp-repression experiments, doxycycline (Sigma-Aldrich) was added to the over-night culture and to the back-dilution media at a final concentration of 15 μg/ml.