Generating Cx30.3 Fusion Constructs

The human Cx30.3-BFP expression vector driven by a CMV promoter was custom ordered from VectorBuilder (Chicago, IL, United States). Employing XhoI and HindIII restriction sites, the BFP tag was replaced by fusing Cx30.3 in-frame to mox-GFP (AddGene, Watertown, MA, United States, Cat# 68070). Plasmids encoding GFP-tagged Cx30.3 mutants (G12D, T85P, and F189Y) were special ordered and purchased from NorClone Biotech Laboratories (London, ON, Canada) and confirmed by sequencing. Red fluorescent protein (RFP) tagged Cx30.3, Cx26, Cx30, and Cx43 were described earlier (Berger et al., 2014 (link); Au et al., 2020 (link); Beach et al., 2020 (link)). The myc-DDK (FLAG)-tagged Cx30.3 expression vector driven by a CMV promoter was purchased from Origene (Rockville, MD, United States, Cat# RC204406). This plasmid was outsourced to NorClone Biotech Laboratories (London, ON, Canada) for the generation of the G12D, T85P, and F189Y mutants via site-directed mutagenesis. Resulting plasmids encoding Cx30.3 mutants were verified by sequencing.