Immunoaffinity purification (using anti-EGFP antibodies) of ribosomes in cells expressing EGFP-L10a was confirmed by detecting a different ribosomal protein, L7 (Figure 1D). After inducing EGFP-L10a INS-1 cells with Dox, we treated them with the ER stress agent thapsigargin (Tg), which inhibits the SERCA (Sarcoplasmic-Endoplasmic Reticulum Calcium ATPase) pump, at a concentration (1 μM) known to trigger apoptosis in the entire population by 24 hours (Han et al., 2009). To reveal proteins translated very early under this regime of irremediable ER stress, we treated the cells with Tg for just 30 minutes before isolating mRNA from either immunoaffinity-purified poly-ribosomes, or total mRNA; both sources of RNA were used to perform comparative DNA microarray analysis (Figure 1E). Validating our approach, Ddit3, a pro-apoptotic UPR transcription factor also known as CHOP, was identified in the hit list of 38 genes with a two-fold or greater change in expression in both total and affinity-purified mRNA (Table S1). CHOP is known to be both transcriptionally and translationally upregulated in the UPR (Jousse et al., 2001; Palam et al., 2011).
