Cells that expressed (A) FKBP-YN and FRB-YC or (B) FKBP-VyN and FRB-YC were analyzed either without pre-treatment (virgin), after 3 days of culture with 25 ng/ml tunicamycin (TM) or after 3 days of culture with tunicamycin followed by 3 days of culture without tunicamycin (TM + rest). An hour before rapamycin induction, the cells were treated with 1 μg/ml tunicamycin (TM), 50 ng/ml thapsigargin (TG), 25 μg/ml anisomycin (Aniso), or 50 μg/ml cycloheximide (CHX). The cells were harvested 3 hours after rapamycin induction and analyzed by flow cytometry. The bar graph shows the mean ratio of BiFC to CFP fluorescence. (C) Diagram depicting the experimental protocol used in parts A and B. The arrows indicate splits of the cells and the X indicates the time plasmids were transfected into the cells.