Journal: International Journal of Biological Macromolecules

Article Title: Proteomic screening identifies RPLp2 as a specific regulator for the translation of coronavirus

doi: 10.1016/j.ijbiomac.2023.123191

Figure Lengend Snippet: Direct interaction between RPLp2 and eIF4E. (A) HEK293T cells were transfected with plasmid to express FLAG-eIF4E or FLAG-eIF4E(S209A) or co-transfected with RPLp2 plasmid. Cell lysates were then immunoprecipitated with FLAG antibodies. The immuno-precipitates were analyzed by Western blot with the antibody to GFP. The expression of proteins from the transfected plasmids was analyzed by Western blot with the indicated antibodies, with β-actin as a control (WCL). (B) Recombinant His-fusion eIF4E and RPLp2 protein were mixed and passed over a Superdex G200 column. Inset: 10 % SDS-PAGE analysis of the marker and peak proteins. (C) Recombinant eIF4E and RPLp2 were mixed in the presence of 0 %, 0.1 %, and 0.05 % cross-linking agent glutaraldehyde (GA) for 15 min, and the reaction was terminated by the addition of 200 mM Tris-HCl buffer, pH 8.0. Cross-linking products were analyzed by 10 % SDS-PAGE and Western blot with antibodies to RPLp2. (D) Docking results for RPLp2 (red) with eIF4E (blue) using ZDOCK. Interacting residues are shown in the stick model. (E) Schematic representation of recombinant constructs of WT RPLp2 and mutants. (F) HEK293T cells were co-transfected with plasmids to express FLAG-eIF4E and GFP-RPLp2 or RPLp2 mutants, as indicated in the schematic diagram. Cell lysates were immuno-precipitated with mouse anti-GFP or IgG antibodies and subjected to Western blot with the indicated antibodies.

Article Snippet: To unravel the interaction domains between eIF4E and RPLp2, eIF4E (PDB no. 5EI3 ) docking into RPLp2 (PDB no. 4BEH ) was performed using the ZDOCK algorithm in Discovery Studio 2018 (Accelrys, San Diego, CA, USA).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Control, Recombinant, SDS Page, Marker, Construct