Figure 4.
Identification of the key binding domain in TrkB interacting with CPE that mediated activity-dependent surface TrkB recruitment. A, Coimmunoprecipitation was performed in HEK 293 cell expressing Myc-CPEC25-RFP and Flag-TrkB mutants. Cell lysates were immunoprecipitated using anti-Flag antibody, followed by immunoblotting with Myc antibodies, and Myc-RFP is a control protein to confirm RFP couldn't combine with TrkB. B, Co-IP of Myc-CPEC25 and FLAG-TrkB deletion mutants as indicated in HEK 293 cells. C, Summary of TrkB mutants used in B and a summary of the CPEC25 interactions with the mutants of TrkB. FL, full-length domain; EX, Extracellular domain. D, Co-IP of Myc-CPEC25 and Flag-TrkB deletion mutants as indicated in HEK293 cells. E, Summary of TrkB mutants used in D and a summary of the CPEC25 interactions with the mutants of TrkB. A–E, Each co-IP experiment was repeated at least three times, and representative data are shown. F, Representative immunofluorescence images of TrkB mutants or T1 chimeras surface levels in transfected hippocampal neurons before and after glycine treatment. Scale bar, 10 μm. G, Quantification of surface levels of TrkB mutants by the ratio of red to green fluorescence intensity in F (two-way ANOVA with Bonferroni's multiple comparisons test, F(1,12) = 7.75, Ctr-TrkB FL versus Gly-TrkB FL **p = 0.0063; Ctr-TrkBΔBOX1 versus Gly-TrkBΔBOX1 *p = 0.0174; Ctr-TrkBΔBOX3 versus Gly-TrkBΔBOX3 *p = 0.0492; n = 4). H, Quantification of surface levels of T1 chimeras by the ratio of red to green fluorescence intensity in F (two-way ANOVA with Bonferroni's multiple comparisons test, F(3,24) = 3.845, ***p = 0.0007; n = 4). All the data are presented as mean ± SEM (n ≥ 25 cells for each condition per experiment).