Dsred er

Canine NCX1.1 cDNA was kindly provided by Professor Godfrey Smith (University of Glasgow, Glasgow, United Kingdom). DsRed-ER was from Clontech (Mountain View, CA, USA) and Grasp65-mCherry was kindly provided by Dr. Jon Lane (University of Bristol, Bristol, United Kingdom). Yellow fluorescent protein (YFP) NCX1 large intracellular loop (YFP-NCX1-IC) was created by inserting canine NCX1.1 cDNA (codons 219 to 761) at the 3′ end of YFP in vector pEFYP-C1 (Clontech). All transfections of plasmid DNA used Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Human embryonic kidney (HEK)-derived FT-293 cells expressing tet-inducible wild-type (WT) and C739A NCX1 were generated using the Invitrogen Flip-In T-Rex system.

α-MSH was purchased from Bachem (Bubendorf, Switzerland). FlAsH-EDT₂ and ReAsH-EDT₂ were either purchased from Invitrogen (Carlsbad, CA, USA) or synthesised in the laboratory of Dr Margaret Brimble (University of Auckland). DsRed-ER, encoding a fusion protein consisting of DsRed with the ER targeting sequence of calreticulin at the amino terminal and the ER retrieval sequence KDEL, at the carboxyl terminal, was purchased from Clontech (Mountain View, CA, USA). Human β-1,4-galactosyltransferase 1 (GalTase) with an N-terminal mCherry tag was generated by subcloning GalTase from peGFP-N1 (GalTase-GFP), which has previously been described [10 (link)], into mCherry-N1, using XhoI and SalI restriction sites and verified by DNA sequencing.