Fig. 1: PKAc interacts with Ptc through Ptc’s intracellular domain C4, which could be regulated by Hh.

Western blot analysis of immunoprecipitates or whole cell lysates from S2 cells expressing indicated constructs including PtcWT-V5 and 3XHA-PKAc (a) or mC*-CFP (b). S2 cells were transfected with the indicated constructs, treated with or without Hh medium, followed by co-Immunoprecipitation with anti-V5/Flg antibodies respectively. Western blot to detect interaction dynamics of SmoWT-PKAc (c) and PtcWT-PKAc (d). Hh-induced interaction reduction between Ptc–PKAc was quantified. e A simple cartoon of Drosophila transmembrane receptor PtcWT which marks its extracellular loops (EL1 and EL4) and intracellular domains (C1, C2, C3, C4, C5, C6, and C7). f S2 cells were transfected with dsRNA against ptc 5′UTR to knockdown endogenous Ptc and co-transfected with indicated expression constructs including 3XHA-PKAc, V5C vector, PtcWT-V5, PtcWTΔC1-V5, PtcWTΔC2-V5, PtcWTΔC3-V5, PtcWTΔC4-V5, PtcWTΔC6-V5, and PtcWTΔC7-V5. Immunoprecipitation assays to detect interaction between PKAc and indicated Ptc variants by western blot. g S2 cells were transfected with the indicated constructs including 3XHA-PKAc, Ptc-C4-V5, and Ptc-C7-V5, followed by co-Immunoprecipitation with anti-V5 antibodies. Western blot to detect interactions between PKAc and Ptc-C4 or Ptc-C7. h S2 cells were transfected with the indicated constructs including 3XHA-PKAc, membrane-tethered Ptc-C4 (Myrflg-Ptc-C4-V5, left half) and membrane-tethered Ptc-C7 (Myrflg-Ptc-C7-V5, right half), followed by co-Immunoprecipitation with anti-HA/Flag antibodies. Western blot to detect interactions between PKAc and Myrflg-Ptc-C4 or Myrflg-Ptc-C7. i GST pull-down assays were performed by using purified His-tagged PKAc (His-PKAc) and GST control or indicated GST-tagged Ptc intracellular domains (GST-C1, -C4, and -C7). Physical interactions of His-PKAc with those GST-tagged peptides were detected by western blot