Fig. 5.
Identification of the ICL3 and CT of DOPr as the major molecular determinants for the interaction with Rab10. (A) Immunoprecipitation of FLAG-DOPr was performed in HEK293 cells transiently expressing FLAG-DOPr and HA-Rab10 using a M2 mouse monoclonal anti-FLAG antibody. (B) In vitro binding assays were carried out using purified GST or the DOPr intracellular loops (ICLs) or C terminus as GST-fusion proteins incubated with purified recombinant (His)6-HA-Rab10. Sequences of the GST-fused protein constructs are listed in SI Appendix, Table S6. Rab10 binding to the receptor was detected by immunoblotting using HA-specific HRP-conjugated antibody, and the GST fusion proteins present in the binding reaction were detected using an anti-GST polyclonal antibody.