Fig. 6. Proneural proteins directly interact with Phyl. (A-C) Western blots of immunoprecipitates (IPs) or lysates from S2 cells expressing the indicated proteins. Immunoprecipitation was performed using anti-Flag antibody. (A) Phyl and Ac proteins interact in S2 cells. Myc-Ac protein was co-immunoprecipitated by anti-Flag antibody only when Flag-Phyl was co-expressed. (B) Myc-Sc protein could only be co-immunoprecipitated by anti-Flag antibody when Flag-Phyl was co-expressed. (C) Phyl and Da do not interact in S2 cells. HA-Da was not co-immunoprecipitated with Flag-Phyl. ^*Non-specific bands. (D,E) GST pull-down assay. (D) GST-Ac and GST-Sc specifically pulled down in vitro translated S³⁵-labelled Phyl and Sina proteins, but not the control Luciferase (Luc) protein. GST protein did not pull down Phyl and Sina. (E) GST-Sina does not interact with Da. As positive controls, GST-Sina pulled down Phyl and Ac. (F) Phyl acts as an adaptor between Ac and Sina in yeast bridge assay. Interaction was scored by the growth in the -His -Ade (plate II) and -His -Ade -Met (plate III) selective plates. All yeast cells grow in the non-selective plates (plate I). Expression of the bridge protein was induced in the absence of 1 mM methionine (-Met).