Figure 2
CC1-EGFP blocked TrkB anterograde axonal transport in cultured hippocampal neurons. (a,b) HEK293 cells were co-transfected with Myc-KLC1, FLAG-TrkB and CC1-EGFP. Lysates were immunoprecipitated with the anti-FLAG antibody. Immunoblotting indicated that CC1-EGFP partially blocked the TrkB-KLC1 interaction. Data are shown as the mean±S.E.M. from three independent experiments (n=3, **P<0.01 compared to the Flag-TrkB-positive and CC1-EGFP-negative group). (c,d) DIV3 hippocampal neurons were co-transfected with GFP or CC1-EGFP and TrkB-mRFP. The kymograph of TrkB-mRFP at the axon was captured every 1 s for 60 s. Scale bar, 20 μm. The relative frequency of retrograde (Retro), anterograde (Antero), bidirectional (Bidir), and immobile TrkB-mRFP puncta at the axons and dendrites was analyzed. The numbers of cells analyzed for each group was>60. Data are shown as the mean±S.E.M. of three independent experiments (n=3; *P<0.05 compared to the EGFP group). (e,f) CC1-EGFP decreased the distribution of endogenous TrkB at the axonal tips. Cultured hippocampal neurons were electroporated with EGFP, siJIP3, or CC1-EGFP, and endogenous TrkB was stained with the anti-TrkB antibody (red) at DIV3. Scale bar, 10 μm. The intensity of TrkB at distal axons and dendrites was analyzed. Data are shown as the mean±S.E.M. from three independent experiments,>60 neurons per experiment (n=3; *P<0.05; compared to EGFP group). (g,h) Neurons were electroporated with EGFP, siJIP3, or CC1-EGFP and were stained for endogenous JNK3 at DIV3. Scale bar, 10 μm. The intensity of endogenous JNK3 at the distal axons and dendrites was analyzed. Data are shown as the mean±S.E.M. of three independent experiments, >60 neurons per experiment (n=3; *P<0.05 compared to the EGFP group)
