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Fig. 8. Glucose and PKA activation enhance the binding activity of CREM1 to the human Cx36 CRE motif in INS-1 cells. Electrophoretic mobility shift assays (EMSAs) were performed using the Cx36 CRE and nuclear extracts (NE) from INS-1 cells cultured at either 2 mM or 20 mM glucose with or without forskolin or H89. For the competition assay, a 100-fold excess of the unlabelled oligonucleotides were used (cold competitor x100). mut, mutated Cx36 CRE; wt, wild-type Cx36 CRE. Compared with the free probe, two slow-migrating complexes (C1 and C2) were detected. The levels of binding are increased in presence of high levels of glucose or forskolin and reduced by H89 treatment. For the supershift analysis, nuclear extract from INS-1 cells cultured at either 2 mM or 20 mM glucose were exposed to specific antibodies directed against CRE modulator protein 1 (CREM-1), activating transcription factor 3 (ATF3), CCAAT/enhancer-binding protein ß (C/EBPß), phosphorylated CRE-binding protein (P-CREB), RE-1-silencing transcription factor (REST, also known as NRSF). CREM-1 and P-CREB supershifted the C1 complexes. Data are representative of four independent experiments.

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