Molecular Tools for TMIGD1 Characterization

Moesin-GFP, ezrin-GFP, GST-FERM-moesin, GST-C-ERMAD-moesin, and LifeAct7 plasmids were purchased from Addgene. Moesin sgRNA was purchased from Dharmacon. Construction and cloning of TMIGD1 into retroviral, pMSCV.puro vector was previously described [2 (link), 27 (link)]. GST-TMIGD1 and GST-4A-TMIGD1 were generated by standard PCR amplification of the cytoplasmic region of TMIGD1 and cloned into pGEX-4T-2 vector. Plasmids were expressed in E.coli and GST-fusion proteins were purified from E.coli via glutathione Sepharose Fast-flow resin per manufacturer’s recommendations. Polyclonal rabbit anti-α-tubulin antibody, polyclonal rabbit-anti-acetyl (K40)-tubulin antibody, anti-Myc antibody, and anti-GFP antibody all were purchased from Cell Signaling Technology Inc., (Danvers, MA). Development and characterization of the polyclonal anti-TMIGD1 antibody used in these studies has been described previously [2 (link)].