Ambion mmessage mmachine sp6 transcription kit

mASIC1a cDNA, cloned between the BamHI and SacI restriction sites of the pSP64 vector, was a gift from Dr. Marcelo Carattino (University of Pittsburgh, Pittsburgh, PA). hENaC α, β, and γ subunits in the pTNT vector were a gift from Dr. Diego Alvarez de la Rosa (University of La Laguna, San Cristóbal de La Laguna, Spain). The α subunit contained the T334 and A663 polymorphisms. The construct referred to as WT hENaC contains C-terminal truncations in the β and γ subunits (β_R566STOP and γ_K576STOP) to increase expression. Site-directed mutagenesis was performed using custom-designed primers (Eurofins Genomics) and regular PCR with PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies). Plasmids were linearized with EcoRI (for mASIC1a) and BamHI (for hENaC) and used as a template for synthesis of mRNA with the Ambion mMESSAGE mMACHINE SP6 Transcription Kit (Thermo Fisher Scientific). ENaCα-mASIC1aM1 chimera DNA cloned between the NheI and Xhol restriction sites in the pUNIV vector was purchased from Twist Bioscience.