mfsd2a  (Addgene inc)

Journal: Science Advances

Article Title: Inhibition of placental trophoblast fusion by guanylate-binding protein 5

doi: 10.1126/sciadv.adt5388

Figure Lengend Snippet: ( A and B ) Syncytin cleavage in the presence of GBPs. Top illustrates the topologies of Syncytin-1 and Syncytin-2, including their polybasic cleavage sites. To monitor Syncytin cleavage, HEK293T cells were cotransfected with Syncytin expression plasmids and increasing amounts of GBP expression plasmids. Two days posttransfection, cells were harvested, and cleavage was analyzed by Western blotting. Syncytin maturation was determined by calculating the amount of cleaved to total Syncytin. Representative Western blots are shown in the middle. Purple arrowheads indicate the shift in the electrophoretic mobility of Syncytin-1. Bottom shows quantifications of three independent Western blots ± SD. SP, signal peptide; TMD, transmembrane domain; CYT, cytoplasmic tail; V5, V5 tag. ( C and D ) Syncytin-mediated cell fusion in the presence of GBPs. HEK239T cells stably expressing the N-terminal portion of GFP were cotransfected with expression plasmids for Syncytin-1 or Syncytin-2 and increasing amounts of plasmids expressing GBP2 (C) or GBP5 (D) and mixed with HEK293T cells expressing C-terminal part of GFP. GFP fluorescence was quantified over a period of 48 hours as a marker for syncytia formation. Mean values of four to eight independent experiments ± SD are shown. ( E and F ) Infectivity of Syncytin-carrying pseudovirions produced in the presence of GBPs. MFSD2A knockout HEK293T cells were cotransfected with plasmids for Syncytin-1 or Syncytin-2 together with ASLV gag - pol , mScarlet minigenome, and GBP or IFITM3. Two days later, supernatants were collected and used for the infection of HEK293T cells overexpressing MFSD2A . Three days postinfection, the percentage of infected cells was analyzed by flow cytometry. IFITM3 served as a positive control. Mean values of three independent experiments ± SD are shown. A one-way ANOVA with multiple comparisons (Dunnett’s test) was performed (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: The coding sequence of MFSD2A ( NP_116182.2 ) was cloned into a previously described gamma-retroviral vector and fused to the coding sequence of GFP ( ). pMD2.G, pMDLg/pRRE, and pRSV rev have been described before ( ) and were obtained through addgene (catalog nos.

Techniques: Expressing, Western Blot, Stable Transfection, Fluorescence, Marker, Infection, Produced, Knock-Out, Flow Cytometry, Positive Control