determination of luciferase mRNA expression, 1 mg of total RNA was reverse-transcribed in a 20 ml reaction mixture by using a Reverse Transcription System (Promega) and random primers following the manufacturer’s instructions. The LightCyclerH FastStart DNA MasterPLUS SYBR Green I system (Roche) was used for real-time PCR amplification/quantification using 2 ml of first strand cDNA reaction as a template. The level of GAPDH mRNA in each sample was determined in order to normalize for differences in total RNA amounts. The data were derived from at least three independent reverse-transcription reactions and real- time PCR performed in duplicate. Data analysis to determine relative luciferase mRNA expression was performed according to the 2-DDCT method [49]. Primers were designed to amplify fragments in the open reading frame of luciferase (nucleotides 1343-1540) and GAPDH (nucleotides 493–775). Statistical Analysis All data are reported as means 6 SE of the results obtained from at least three independent experiments. Data were analyzed by two-tailed, unpaired Student’s t test. Differences were considered significant when P,0.05. Supporting Information Figure S1 GCN2 and PKR expression and eIF2alpha kinase response in BHK-C and BHK-A1 cells. (A) BHK-C and BHK-A1 cells were maintained for 1 h in normal growth References 1. Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397: 271–274. 2. de Haro C, Mendez R, Santoyo J (1996) The eIF-2alpha kinases and the control of protein synthesis. FASEB J 10: 1378–1387. 3. Chen JJ, London IM (1995) Regulation of protein synthesis by heme-regulated eIF-2 alpha kinase. Trends Biochem Sci 20: 105–108. 4. Berlanga JJ, Herrero S, de Haro C (1998) Characterization of the hemin- sensitive eukaryotic initiation factor 2alpha kinase from mouse nonerythroid cells. 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