Romanian Biotechnological Letters Vol. 18, No.4, 2013 Copyright © 2013 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER Romanian Biotechnological Letters, Vol. 18, No. 4, 2013 8539 Current methods for investigation of the unfolded protein response Received for publication, March 15, 2013 Accepted, July 15, 2013 ALINA STROESCU, LIVIA SIMA, FLORENTINA PENA* Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 060031 Bucharest 17, Romania. * Corresponding author (Email: Florentina.Pena@biochim.ro; phone: +40212239069; fax +40212239068) Abstract The endoplasmic reticulum (ER) is the cellular organelle where proteins from the secretory pathway fold. The ER is a very dinamic organelle that can respond rapidly to changes in the ER load by upregulating the ER resident proteins that are involved in the folding process. Simultaneously, less proteins from the secretory pathway are synthesized. The whole cascade of events is called the unfolded protein response (UPR). The UPR can originate from physiological or pathological conditions and many times the ER changes in a subtle manner. Researchers working in the ER field developed various tools to assay the ER stress. Here we describe methods optimized in our laboratory and show that western blotting, quantitative real time polymerase chain reaction (RT-qPCR) and reverse transcription-polymerase chain reaction (RT-PCR) are reliable, cost-effective techniques that researchers can choose to evidence ER stress activation. Abbreviations: BiP, immunoglobulin heavy-chain binding protein; ER, endoplasmic reticulum; TG, thapsigargin; TN, tunicamycin; UPR, unfolded protein response; IRE1, inositol-requiring kinase 1; XBP1, X-box- binding protein 1; PERK, RNA-dependent protein kinase-like ER kinase; ATF6, activating transcription factor 6; eIF2 , eukaryotic translational initiation factor 2 Bcl-2, B cell lymphoma 2, ATF4, activating transcription factor 4; CHOP, CCAAT/enhancer-binding protein-homologous protein; GRP, glucose-regulated proteins, CREB-RP, cyclic AMP-response-element-binding protein- related protein; S1P, site-1 protease;S2P, site-2 protease; ERSE, ER stress response elements; Grp94, Glucose-regulated protein 94; ERp44, endoplasmic reticulum resident protein 44 kDa; ERp72, endoplasmic reticulum resident protein 72 kDa; ERp57, endoplasmic reticulum resident protein 57 kDa; Ero1 , ER oxidoreductin 1 ; DTT , dithiothreitol; GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase; ERAD, ER-associated degradation; ECL, enhanced chemiluminescence; RT-PCR, reverse transcription-polymerase chain reaction; RT-qPCR, quantitative real time polymerase chain reaction. 1. The endoplasmic reticulum – a short overview The endoplasmic reticulum (ER) is essential for production of secretory proteins (BRAAKMAN [1]). Proteins from secretory pathway emerge from ER-bound ribosomes and are further modified and folded by the ER folding machinery. There are three major classes of ER resident proteins: molecular chaperones, foldases and lectins (SCHRODER [2]). The molecular chaperones facilitate protein folding by shielding unfolded regions from surrounding proteins. BiP for instance binds to the hydrophobic patches that are exposed during folding, thus preventing aggregation. The foldases (cis–trans peptidyl–prolyl isomerases, PPI/immunophilins and PDIs) catalyze steps in protein folding such as the formation and isomerization of disulfide bonds. Lectins interact with glycoproteins due to the presence of a lectin site that can recognize an early folding oligosaccharide processing intermediate on the folding glycoprotein (WILLIAMS [3]). Correctly folded secretory proteins are exported to intracellular organelles or to the extracellular surface. Proteins