Downstream of tyrosine kinase/docking protein (Dok)6 binds to tropomyosin-related kinase (Trk)C receptor in glutathione S-transferase (GST) precipitation and coimmunoprecipitation (Co-IP) assays. (a) The Dok6 phosphotyrosine-binding (PTB) domain binds to the TrkC receptor in GST precipitation assays. Human embryonic kidney (HEK)293 cells were transfected with pcDNA3.1V5-Trks and treated with or without neurotrophins (100 ng/ml) for 10 min. The cell lysates were incubated with Dok6-PTB-GST Sepharose 4B beads at 4°C for 2 h. The bound proteins were then analysed by western blotting with anti-V5 antibody (upper panel). Cell lysates were examined by anti-V5 or anti-pTyr antibodies to detect the expression and phosphorylation levels of Trk receptors (second and third panel). Dok6-PTB-GST proteins were tested by Coomassie Brilliant Blue staining (bottom panel). (b) Dok6 binds to the TrkC receptor in Co-IP assays. HEK293 cells were cotransfected with Flag-Dok6 and V5-TrkC. Before harvest, the cells were treated with or without 100 ng/ml NT3 for 10 min. Cell lysates were immunoprecipitated by anti-Flag and then were detected by western blotting with anti-V5 and anti-Flag.
Li et al. BMC Biology 2010 8:86 doi:10.1186/1741-7007-8-86