Strategy for investigating the translational efficiency of mRNAs by polysome profiling and microarray hybridization. A representative polysome profile shows the monosome peak (1) at the top of the gradient (left), followed by peaks representing 2,3,4 and ≥ 5 ribosomes per mRNA. Following centrifugation, the gradient was divided into two: an under-translated fraction containing mRNAs associated with 1-4 ribosomes (fraction-U) and a well translated fraction containing mRNAs associated with 5 or more ribosomes (fraction-W). The mRNAs in each fraction were then used to interrogate microarrays, as detailed in Methods. The translational efficiency of each mRNA was defined as the ratio of the hybridization signal (fraction-W/fraction-U). Those mRNAs with a higher W/U ratio during ER stress than in the absence of ER stress (using a 2-fold change between conditions as the cut-off) were considered to be subject to translational upregulation during ER stress.