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. 2014 May;88(10):5816–5833. doi: 10.1128/JVI.03826-13

FIG 3.

FIG 3

Experiments showing the interaction between EV71 VP1 protein and vimentin. (A) Detection of binding between VP1 and vimentin using GST pulldown assays and Western blotting. The eluates obtained as described in Materials and Methods were blotted with antivimentin and anti-GST antibodies, and the resultant VP1 protein band observed is indicated. Input, untreated cell lysate; control, U251 cell lysate incubated with glutathione-Sepharose beads; VP1, 3C, and GST, U251 cell lysate incubated with VP1-GST, 3C-GST, or GST precombined glutathione-Sepharose beads, respectively. (B) Pulldown assays and Western blot analysis showing the interaction between VP1 and vimentin. GST or VP1-GST protein was incubated with antivimentin monoclonal antibody-conjugated agarose beads that were preincubated with vimentin protein. The precipitated proteins were blotted with antibodies to either vimentin or GST, and the resultant VP1-GST protein band observed is indicated. (C) Analysis of the binding of VP1 to vimentin by coimmunoprecipitation and Western blotting. U251 cells were transfected with a plasmid expressing VP1, lysed, and coimmunoprecipitated with vimentin antibodies (anti-Vim) or mouse IgG. The precipitated proteins were blotted with antibodies to vimentin and EV71 VP1. Input, cell lysate; control, agarose beads incubated either with no antibodies or with IgG; anti-Vim and mouse IgG, agarose beads incubated with vimentin antibody and mouse IgG, respectively. (D) Coimmunoprecipitation and Western blot analysis of the binding of vimentin to VP1, VP2, and VP3. U251 cells were transfected with plasmids expressing either VP1, VP2, or VP3. Coimmunoprecipitation was performed with Flag antibody. The precipitated proteins were analyzed by Western blotting using antibodies to vimentin and Flag. Control, precipitated proteins from cells with no plasmid transfection; VP1, VP2, VP3, precipitated proteins from cells transfected with VP1, VP2, VP3 plasmids, respectively. The figure shows only EV71 VP1 interacted with vimentin and not VP2 or VP3.