Figure 2.

Downregulation of ephrinA5-evoked increase in retinal axon branching via RNAi-mediated knockdown of TrkB. A, CHO cells were transfected with TrkB^FLAG alone, with TrkB^FLAG and the empty RNAi vector, or with one of three different RNAi specific for TrkB. Lysates were analyzed the following day for TrkB expression levels, which were normalized by blotting with an α-tubulin antibody. All three RNAi let to a significant downregulation of TrkB expression. RNAi with point mutations in the TrkB targeting sequence (e.g., RNAi-4**; see Materials and Methods) had no effect on TrkB expression level (right). B, Abolishment of BDNF-mediated increase in retinal axon branching by expression of three different TrkB RNAi. The summary from three independent experiments is shown. Statistical analysis was performed using one-way ANOVA with Tukey's multiple-comparison test. C, The increase in branching mediated by ephrinA5 overexpression (see also Fig. 1) and its further increase by BDNF application can be diminished by overexpression of TrkB RNAi. The downregulation of branching is not complete, indicating that the knockdown of TrkB is not complete or that other molecules are involved in mediating the effect of ephrinA5 overexpression on branching. A reduction in branching is apparent also in the absence of BDNF, suggesting that overexpression of ephrinA5 results in a ligand-independent TrkB/ephrinA5 interaction. The summary from three independent experiments is shown. Statistical analysis was performed using one-way ANOVA with Tukey's multiple-comparison test. *p = 0.05; **p = 0.01; ***p = 0.001.