FIG 5.

Role of Ehmeth Cys228 and Cys229 in the formation of the Ehmeth-enolase complex. (A) Western blot analysis of recombinant proteins (Ehmeth, Ehmeth C228S, Ehmeth C229S, and Ehmeth C228S-C229S) that were treated with 5 μM GSNO for 1 h at 37°C. The proteins were resolved on 12% polyacrylamide gels under native conditions, transferred to a nitrocellulose membrane, and then probed with an S-NO-Cys antibody (α S-NO-Cys). Ponceau staining of the membrane prior to its interaction with the S-NO-Cys antibody was used as a loading control. The Ehmeth plus DTT control shows the results with the Ehmeth recombinant protein treated with 5 μM GSNO for 1 h at 37°C followed by incubation with 20 mM DTT for 5 min at 37°C. The figure displays a representative result from at least three independent experiments performed singly. (B) Western blot analysis of nuclear protein fractions prepared from pJST4-Ehmeth and pJST4-Ehmeth C228S-C229S E. histolytica trophozoites performed using an HA antibody, an enolase antibody, or an actin antibody. The figure displays a representative result from at least three independent experiments performed singly. (C) Immunoprecipitation analysis of Ehmeth from pJST4-Ehmeth and pJST4-Ehmeth C228S-C229S E. histolytica trophozoites, performed with an HA antibody. The presence of enolase among the immunoprecipitated proteins was detected by using an enolase antibody. The amounts of Ehmeth and Ehmeth C228S-C229S in the pJST4-Ehmeth and pJST4-Ehmeth C228S-C229S E. histolytica trophozoites were determined by using a histidine antibody.