Generation of mitochondrial cAMP sensor

The mitochondrial-targeting sequence 4mt, encoding four copies of the signal sequence from subunit VIII of human cytochrome C oxidase, was amplified using the Advantage Polymerase (Clontech, Mountain View, CA, USA) and primers F, 5′-ACTATAGGGAGACCCAAGCTTATG-3′ and R, 5′-TGGTGGCGGCAAGCTTCTTGCTCACCATGGTGGC-3′. The pcDNA-4mt-D3-cpv vector used as a matrix for amplification of 4mt was a kind gift from Dr. Roger Tsien (HHMI investigator at the University of California San Diego, San Diego, CA, USA). The PCR fragment was cloned into the HindIII restriction site of pcDNA3-Epac-S^H187 using the Infusion HD Cloning System (Clontech). Epac-S^H187 encodes for a fourth-generation Epac1-based cAMP sensor and was a kind gift from Dr. Kees Jalink (The Netherlands Cancer Institute, Amsterdam, Netherlands).^{36 (link)} Once the pcDNA-4mt-Epac-S^H187 vector was amplified in Stellar Escherichia coli (Clontech) bacteria, its identity with parental sequences was verified by PCR using primers F, 5′-ACTCACTATAGGGAGACC-3′ and R, 5′-TGCGGCCGCCATGGTGGC-3′, and DNA double-strand sequencing (INSERM U1056 – UMR 5165 CNRS UPS – UDEAR, Toulouse, France). Adenoviruses encoding Epac-S^H187 and 4mt-Epac-S^H187 were generated by Welgen Inc (Worcester, MA, USA).