on ABI Prism 7500 sequence detector (Applied Biosystems Inc, Foster City, CA, USA). Primers used in the experiments were: Human PEPCK: 59- CATAAAGGCAAAATCATCATGCA (sense), 59- TTGCCGAAGTTGTAGCCAAA (antisense); Human HSP70: 59-TGTCGTCCAGCACCCAGGCCAGC (sense), 59- GCTCTTGTTCAGGTCGCGCCCG (antisense). Human 36B4: 59-TGCTGAACATGCTCAAC (sense), 59-GTCGAACACCTG- CTGGATGAC (antisense). Amplification of the PEPCK or HSP70 cDNA was normalized to 36B4 expression. Relative levels of target mRNA expression were calculated using the 2-DDCT method. Immunoblotting Cell lysate was prepared with cold RIPA lysis buffer as previously reported [26]. Nuclear extract was prepared by NE- PERTM Nuclear and Cytoplasmic Extraction Reagents (Thermo, Rochford, USA) according to the manufacturer’s instructions. The protein concentration was determined using Bradford assay. 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Wang O, Cai K, Pang S, Wang T, Qi D, et al. (2008) Mechanisms of glucose- induced expression of pancreatic-derived factor in pancreatic beta-cells. Endocrinology 149: 672–680. PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13096