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. 2011 Jul 20;31(29):10602–10614. doi: 10.1523/JNEUROSCI.0436-11.2011

Figure 5.

Figure 5.

Mapping the JIP3 binding domain with TrkB. A, Coimmunoprecipitation of TrkB-FL with JIP3 mutants. HEK293 cells were cotransfected with FlagTrkB-FL and HAJIP3 mutants, and immunoprecipitation was performed using rabbit anti-HA antibodies, immunoblotted with mouse anti-Flag or mouse anti-HA antibodies. B, Coimmunoprecipitation of KLC1 with JIP3 mutants. HEK293 cells were cotransfected with MycKLC1 and HAJIP3 mutants, and immunoprecipitation was performed using rabbit anti-HA antibodies, immunoblotted with mouse anti-Myc or mouse anti-HA antibodies. C, Schematic representation of the HAJIP3 mutants. Amino acid numbers correspond to appropriate domains within the JIP3 deletion constructs. A summary of TrkB-FL/KLC1 interactions with the JIP3 mutants is provided. D, Interaction of TrkB with KLC1 was reduced by HAJIP3ΔCC1. HEK293 cells were cotransfected with MycKLC1, FlagTrkB-FL, and HAJIP3/JIP3ΔCC1 constructs, and immunoprecipitation was performed using rabbit anti-Myc antibodies, followed by immunoblotting with mouse anti-Myc, anti-Flag, or anti-HA antibodies. E, Direct interaction of the TrkB-JM1 domain with the JIP3-CC1 domain in vitro. GST-JM1, GST-JM2, GST-JM3, and GST-JM fusion proteins were incubated with the purified His-CC1. The precipitate was subjected to Coomassie Brilliant Blue staining and immunoblotting analysis using anti-His antibodies. A summary of JM mutants' interactions with CC1 is provided.