Western blots (WB) showing co-immunoprecipitation using DdS4 antibody; lower panels in C and D show Coomassie-stained proteins.

(A) Cell lysates from cells over-expressing Cdc42p, Cdc24p, Bem1p and Cla4p as GST-tagged proteins were mixed in equal volumes. Subsequently upon specific interaction with DdS4 antibody the proteins were resolved on 12% SDS PAGE and probed with anti-GST antibodies. The proteins resolved on the gel were identified on the basis of their molecular weights. Specific interactions between the GST-tagged proteins and DdS4 are observed in the IP lane. Controls: IgG-pull down using normal rabbit IgG or vector-lysates from cells over-expressing the GST-tagged proteins and an empty vector, show no interactions. (B) Individual cell lysates from E. coli BL21 over-expressing Cdc42p and Cdc24p as GST-tagged proteins respectively were allowed to interact with GST (Glutathione S-transferase) beads subsequently the beads were incubated with a cell lysate over-expressing DdS4. The GST beads were run on 12% SDS PAGE and probed with anti-DdS4 antibody. The input panel shows that equal amount of beads were loaded. (C) and (D) Specific interactions studied using antibodies raised against DdS4 and commercial anti-CDC24 or anti-CDC42 antibodies. (C) Cell lysates from S. cerevisiae bem1 or control AX2 D. discoideum cells were probed with anti-Cdc24 or anti-GxcDD antibody. IgG lane is a control with normal rabbit IgG. IP lane shows specific interaction when anti-DdS4 antibody is used for pull-down. Pt-G lane is the pull down using protein G beads alone. (D) Cell lysates from bem1 or control AX2 cells probed with anti-Cdc42. The lanes are labeled as described above. Coomassie stained of the nitrocellulose blot shows equal loading.