Ezrin gfp

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)].

Primary mouse hepatocytes were isolated and cultivated as described previously (29 (link)). HeLa cells (gift from Robert Menard, Pasteur Institute, Paris, France) were authenticated by short tandem repeat DNA profiling (Microsynth) and cultured as described before (30 (link)). We transfected 2 × 10⁶ HeLa cells with 4 µg of plasmid DNA of the following fluorescent reporter constructs with program T-28 in an Amaxa Nucleofector (Lonza): mCherry-utrophin (7 (link)) (Addgene plasmid 26740), GFP-utrophin (7 (link)) (Addgene plasmid 26737), GFP-C1-PLCdelta-PH (12 (link)) (Addgene plasmid 21179), AKT-PH-GFP (13 (link)) (Addgene plasmid 18836), Lact-C2-GFP (14 (link)) (Addgene plasmid 22853), ezrin-GFP (15 (link)), ezrin(T567D)-GFP (16 (link)) (Addgene plasmid 20681), E-cadherin–GFP (17 (link)) (Addgene plasmid 28009), A2BR-YFP (18 (link)) (Addgene plasmid 37202), GFP-DRD2 (19 (link)) (Addgene plasmid 24099), PM-GFP (20 (link)) (Addgene plasmid 21213), and GFP-GPI (TRAIL and DAF) (21 (link)). Cells were subsequently used to seed glass bottom dishes (MatTek, In Vitro Scientific) and infected the next day with P. berghei sporozoites as previously described (28 (link)).