Journal: Journal of Virology

Article Title: Epstein-Barr Virus Protein EB2 Stimulates Translation Initiation of mRNAs through Direct Interactions with both Poly(A)-Binding Protein and Eukaryotic Initiation Factor 4G

doi: 10.1128/JVI.01917-17

Figure Lengend Snippet: EB2 interacts in vitro with the eIF4G and PABP initiation factors through its C-terminal domain. (A) Schematic representation of both recombinant GST-EB2 fusion proteins and the eIF4G plus eIF4G deletion mutants used. (B) GST pulldown assays were performed using GST, GST-EB2, GST-EB2Nter, or GST-EB2Cter and [35S]methionine-radiolabeled proteins eIF4GNter, eIF4Gp100, PABP, or eIF4E, synthesized in vitro in a rabbit reticulocyte lysate (RRL), under untreated or RNase-treated conditions as indicated. (C) GST pulldown assays were also performed with GST or GST-PABP fusion proteins and [35S]methionine-radiolabeled proteins EB2 (full length), EB2Nter, or EB2Cter, synthesized in vitro in RRL, under untreated or RNase-treated conditions. Eluates were subjected to SDS-PAGE and analyzed by autoradiography. In lane 1, the equivalent of 1/10 of the total RRL used in each assay was loaded onto the gel as an input control.

Article Snippet: Immunopurified proteins were then analyzed by Western blotting using anti-Flag (Sigma-Aldrich), anti-PABPC1 (Sigma-Aldrich), anti-eIF4A (Cell Signaling Technology), anti-eIF4GI, anti-eIF4B, and anti-eIF4E (kindly provided by Simon Morley, University of Sussex, UK), anti-eIF2α (Cell Signaling Technology), anti-eIF3 (kindly provided by Pierre Jalinot, LBMC-ENS de Lyon, France), anti-rpS6 (Cell Signaling Technology), anti-BMRF1 (Cell Signaling Technology), anti-rpL11 (Cell Signaling Technology), anti-NXF1, and anti-CBP80 (kindly provided by Elisa Izaurralde, Max Planck Institute for Developmental Biology, Germany).

Techniques: In Vitro, Recombinant, Synthesized, SDS Page, Autoradiography