Binding of EhARPC1 with EhAK1.

(A) Co-precipitation of EhCaBP1 with GST-tagged protein (GST-EhARPC1 or GST-EhC2PK) was tested using Glutathione Sepharose beads. Beads were first loaded with recombinant GST-tagged indicated proteins and then incubated in presence of recombinant EhCaBP1. Beads were washed, and eluted proteins were detected using anti-EhCaBP1 antibody in western blots. (B) Co-immunoprecipitation of EhCaBP1 from whole amoebic lysate was done using agarose conjugated with either anti-EhARPC1 antibody or pre-immune serum (PB). The beads were washed and the eluted proteins were probed by western blot analysis using indicated antibodies. (C) Co-precipitation of his-tagged EhARPC1 with GST-tagged EhC2PK and GST-tagged EhAK1 was carried out in similar way as mentioned in (b), proteins were detected with anti-his antibody. EhAK1 was able to precipitate his-tagged EhARPC1 whereas neither GST EhC2PK nor GST alone was able to precipitate his-tagged EhARPC1. (D) Immunoprecipitation of EhARPC1 from whole-cell lysate of E. histolytica using CNBr-conjugated anti-EhAK1 antibody. The total input lysate was used for the presence of EhARPC1 and EhAK1 using respective antibodies. (E) Schematic diagram showing the organization of EhAK1 (KD and SH3) and EhARPC1 (N-ter and C-ter) constructs. (F) Co-precipitation of his-tagged EhAK1, KD and SH3 with GST-EhARPC1, N-ter and C-ter was carried out in a similar way as mentioned above in (b), only EhARPC1 and N-ter was able to pull SH3 domains. All SDS-PAGE was carried out using 10%-12%polyacrylamide gels unless otherwise mentioned.