Tin Transcription Factor Activation Assay

Co-transfection assays were carried out essentially as described by Kelly Tanaka et al. (2008) (link). The WT tin expression plasmid was pPAc-tin (Lovato et al., 2015 (link)). The tin^R321N mutant expression plasmid was generated using a Q5 site-directed mutagenesis kit (New England Biolabs). The reporter plasmid was the Mp3D cardiac enhancer from the Mp gene (Lovato et al., 2023 (link) preprint) fused to lacZ in the parent vector pDONR lacZ attB (Bryantsev et al., 2012 (link)). Reporter expression was assessed using a mammalian β-galactosidase assay kit (Pierce Technology, Thermo Fisher Scientific). Reporter expression in the presence of transcription factors was normalized to reporter expression in the presence of empty expression vector. Percentage activation is defined as the level of reporter activity in the presence of the activator (WT Tin or Tin^R321N), divided by the level of reporter activity in the absence of an activator (set to 100%). Ten replicates were carried out, and the reporter activities were analyzed using one-way ANOVA and post-hoc Tukey tests.