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

Figure 3.

Figure 3.

Ternary complex formation of JIP3, TrkB, and KLC1 in vitro and in vivo. A, Coimmunoprecipitation of JIP3 with TrkB-FL/TrkB.T1. Lysates from HEK293 cells transfected with HAJIP3 and Flag-tagged TrkB-FL/TrkB.T1 constructs were immunoprecipitated with anti-HA antibodies or anti-Flag antibodies. Then, immunoblotting analysis was performed to detect immunoprecipitated proteins. B, Endogenous JIP3 associates with TrkB and KLC1. Rat brain lysates were subjected to immunoprecipitation with mouse anti-JIP3 or goat anti-KLC1 antibodies, and the protein complex was eluted; the TrkB, KLC1, and JIP3 were detected by immunoblotting. C, JIP3-dependent interaction between TrkB and KLC1. Lysates from HEK293 cells cotransfected with MycKLC1 and FlagTrkB-FL/FlagTrkB.T1 constructs were immunoprecipitated with anti-Myc antibodies. Then, immunoblotting analysis was performed to detect immunoprecipitated proteins. For FlagTrkB-FL: TrkB-FL wild-type group, cotransfected with FlagTrkB-FL and MycKLC1, with endogenous JIP3 expression; JIP3 overexpressed group, cotransfected with FlagTrkB-FL, MycKLC1, and HAJIP3; siJIP3 group, cotransfected with FlagTrkB-FL, MycKLC1, and siJIP3. For FlagTrkB.T1: TrkB.T1 wild-type group, cotransfected with FlagTrkB.T1 and MycKLC1, with endogenous JIP3 expression; siJIP3 group, cotransfected with FlagTrkB.T1, MycKLC1, and siJIP3. Quantitation of immunoblotting in the top panel of Western blots in C is shown as a percentage of TrkB coimmunoprecipitation by KLC1 in different groups compared with TrkB-FL wild-type group. Data are shown as the mean ± SEM from three independent experiments [n = 3; *p < 0.05, vs FlagTrkB-FL wild-type (WT) group; ##p < 0.01, vs FlagTrkB.T1 WT group, one-way ANOVA]. D, The KLC1-independent interaction between TrkB-FL and JIP3. Lysates from HEK293 cells transfected with HAJIP3 and FlagTrkB-FL constructs were immunoprecipitated with anti-HA antibodies. Then, immunoblotting analysis was performed to detect immunoprecipitated proteins. Wild-type group, Cotransfected with FlagTrkB-FL and HAJIP3, with endogenous KLC1 expression; KLC1 overexpressed group, cotransfected with FlagTrkB-FL, MycKLC1, and HAJIP3; siKLC1 group, cotransfected with FlagTrkB-FL, HAJIP3, and siKLC1. E, Simultaneous knockdown of JIP3 and Rab27B further decreases the association between TrkB-FL and KLC1. Lysates from HEK293 cells cotransfected with MycKLC1 and TrkB-FL constructs were immunoprecipitated with anti-Myc antibodies. Wild-type group, Cotransfected with FlagTrkB-FL and MycKLC1, with endogenous JIP3 and Rab27B expression; siJIP3 group, cotransfected with FlagTrkB-FL, MycKLC1, and siJIP3; siRab27B group, cotransfected with FlagTrkB-FL, MycKLC1, and siRab27B; siJIP3+siRab27B group, cotransfected with FlagTrkB-FL, MycKLC, siJIP3, and siRab27B. Quantitation of immunoblotting in the top panel of Western blots in E is shown as a percentage of TrkB coimmunoprecipitation by KLC1 in different groups compared with wild type. Data are shown as the mean ± SEM from three independent experiments (n = 3; *p < 0.05; **p < 0.01, vs the FlagTrkB-FL WT group; #p < 0.05, vs siJIP3+siRab27B group; one-way ANOVA). IP, Immunoprecipitation; CO-IP, coimmunoprecipitation.