Fig. 5.

Phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB) bound mcu gene promoter regions in the spinal cord dorsal horn (SCDH) and mediated mitochondrial calcium uniporter (MCU) transcription. (a) We analysed the alignment of rat MCU gene promoter regions, and found two putative CRE-binding areas, Site 1 (TGACGTAA)²⁹ and Site 2 (TGAGGTCA),30, 31 and chromatin immunoprecipitation with quantitative PCR (ChIP-qPCR) primer areas of the MCU gene at rat chromosome 20 (accession number: NC_005119; NCBI Reference Sequence: NC_005119.4). The TATA box of mcu gene was shown. The first base C (chr 20: 29199224) of the first intron of the mcu gene was referred to as number +1. (b) Motif of the CREB-binding sites based on the JASPAR data sets (http://jaspar.genereg.net/matrix/MA0018.2). (c and d) ChIP-qPCR assay showed that enrichment of pCREB at CRE Site 1 or 2 of the mcu gene promoter region in the mismatch oligodeoxynucleotide (mmODN)+morphine anti-nociceptive tolerance (MT) group was increased compared with the mmODN+sham group (*P<0.05; one-way analysis of variance [anova]; n=5). Enrichment of pCREB at Site 1 or 2 on mcu gene promoter in the AS-CREB+MT group was lower than that in the mmODN+MT group (**P<0.01; one-way anova; post hoc Fisher's protected least significant difference [PLSD]; n=5). (e) RT–PCR showed increased MCU mRNA expression (***P<0.001; one-way anova; post hoc Fisher's PLSD test; n=5). (f and g) Western immunoblots showed no differences in expression of MCU protein between mmODN+sham and AS-CREB+sham groups (f). (g) MCU protein expression increased (**P<0.01; one-way anova). (h–j) Double immunostaining showed that pCREB was co-localised with MCU in the SCDH in morphine-tolerant rats; scale bar: 50 μm.