PMC:4576194 / 6861-10563
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4576194","sourcedb":"PMC","sourceid":"4576194","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4576194","text":"2.2. The Domain I of eEF1A is Critical for the NS5A–eEF1A Interaction\nTo determine which domain(s) of eEF1A is responsible for the association with NS5A, a series of Myc-tagged deletion mutants of eEF1A were constructed (Figure 2A) and tested for the interaction with NS5A by Co-IP assay. The results showed that eEF1A(1-133) and eEF1A(1-237) were capable of binding NS5A (Figure 2B), indicating that the domain I is critical for the NS5A–eEF1A interaction.\nFigure 1 Interaction of NS5A and eEF1A. (A) Reactivity of NS5A with eEF1A in a yeast two-hybrid (Y2H) system. Yeast Y2HGold strain was cotransformed with pGBKT7-NS5A (BD-NS5A) as a bait and pGADT7-eEF1A (AD-eEF1A) as a prey. As a positive control, pGBKT7-p53 (BD-p53) and pGADT7-T (AD-T) were cotransformed into Y2HGold strain. Cotransformation of pGBKT7-Lamin C (BD-Lamin C) and AD-T was used as a negative control. All the yeast colonies cotransformed with the above plasmids were grown on synthetically defined (SD) medium lacking His and Leu (SD/-2), SD medium lacking His, Leu, Trp and Ade (SD/-4) and SD/-4 medium containing 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-Gal) and aureobasidin A (Aba) (SD/-4/X-α-Gal/Aba); (B) Glutathione S-transferase (GST) pulldown assay. The GST or GST-NS5A fusion proteins expressed in Escherichia coli BL21(DE3) were purified with glutathione Sepharose 4B resin and incubated with the lysate of HEK293T cells overexpressing the Myc-tagged eEF1A. After washing with cold PBS, the bound proteins were subjected to sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%) and Western blotting using the anti-GST polyclonal antibody (PAb) (1:2000) and the anti-Myc monoclonal antibody (MAb) (1:1000); (C) Coimmunoprecipitation (Co-IP) analysis of Myc-tagged eEF1A and Flag-tagged NS5A. HEK293T cells were cotransfected with the indicated plasmids (+) or empty vectors (-) for 48 h. The transfected cells were lyzed and incubated with a mouse anti-Flag MAb, followed by incubation with the Protein G-Agarose for 6 h at 4 °C. The immunoprecipitate was analyzed by Western blotting using the anti-Flag MAb (1:1000) and a rabbit anti-Myc PAb (1:500); (D) Co-IP analysis of eEF1A and NS5A by the anti-Myc MAb. HEK293T cells were cotransfected with the indicated plasmids (+) or empty vectors (−), the cell lysate was collected at 48 h post-transfection (hpt), incubated with the anti-Myc MAb and Protein G-Agarose. The immunoprecipitate was examined by Western blotting using the anti-Flag MAb (1:1000) and the anti-Myc PAb (1:500); (E) Effects of RNase A/T1 treatment on the NS5A–eEF1A interaction. Extracts of HEK293T cells overexpressing different proteins were treated or untreated with RNase A/T1 for 1 h prior to Co-IP and immunoblotted with the indicated antibodies. The total RNA in the immunoprecipitate was visualized by ethidium bromide-staining; (F) Colocalization of NS5A protein with eEF1A. HEK293T cells were cotransfected with p3×Flag-NS5A and pMyc-eEF1A. Cells were fixed at 48 hpt and subjected to indirect immunofluorescence assay to detect 3×Flag-NS5A (green) and Myc-eEF1A (red) with mouse anti-Flag and rabbit anti-Myc antibodies. The position of the nucleus is indicated by DAPI (blue) staining in the merged image. Samples were imaged and magnified 630 times on the Leica SP2 confocal system fitted with a 63× objective lens. Scale bar = 7.5 μm.\nFigure 2 The domain I of eEF1A is required for its association with NS5A. (A) Schematic representation of eEF1A protein domains and the individual eEF1A deletion mutants tested in this study; (B) Coimmunoprecipitation (Co-IP) analysis of the association of 3×Flag-NS5A with Myc-eEF1A mutants in cotransfected HEK293T cells.\n\n2","divisions":[{"label":"Title","span":{"begin":0,"end":69}},{"label":"Figure caption","span":{"begin":458,"end":3376}},{"label":"Figure caption","span":{"begin":3375,"end":3701}}],"tracks":[]}