Skip to main content
. 2017 Jun 21;37(25):5978–5995. doi: 10.1523/JNEUROSCI.3148-16.2017

Figure 5.

Figure 5.

UCH-L1 regulates TrkB deubiquitination. A, TrkB undergoes multimonoubiquitination in response to BDNF. Primary neurons were serum starved for 8–12 h before stimulation with BDNF (50 ng/ml) for 15 min. The ubiquitination of TrkB was examined by immunoprecipitation with anti-TrkB antibodies followed by immunoblotting with P4D1 antibodies, which recognize both multimonoubiquitinated and polyubiquitinated substrates, and FK1 antibodies, which recognize only polyubiquitinated substrates. B, β-catenin was polyubiquitinated. Primary neurons were serum starved for 8–12 h and the ubiquitination of β-catenin was examined by both P4D1 and FK1 antibodies. C, TrkB is not ubiquitinated by K48 or K63 polyubiquitin chains. Primary neurons were serum starved for 8–12 h before stimulation with BDNF (50 ng/ml) for 15 min. The ubiquitination of TrkB was examined by immunoprecipitation with anti TrkB antibodies followed by immunoblotting with P4D1, anti-K48, and anti-K63 antibodies. D, UCH-L1 was knocked down with three siRNAs in primary neurons and the ubiqutination of TrkB was examined. E, UCH-L1 was overexpressed with lenti-WT UCH-L1 or C90S UCH-L1 in primary neurons and the ubiquitination of TrkB and the binding between UCH-L1 and TrkB were examined. F, Quantification of UCH-L1 and TrkB in cultured primary neurons before and after BDNF application. *Significant difference with p < 0.05, Student's t test. G, UCH-L1 deubiquitinates TrkB dependent on its hydrolase activity. HEK293 cells were transfected with Myc-UCH-L1, C90S, or D30A mutants together with Flag-TrkB FL and HA-Ub. Cell lysates were immunoprecipitated using anti-Flag antibodies and analyzed by immunoblotting with HA antibodies. H, UCH-L1 deubiquitinates TrkB in an in vitro deubiquitination assay. HEK293 cells were transiently transfected with Flag-TrkB and HA-Ub and ubiquitinated TrkB was enriched with anti-Flag antibodies. Purified UCH-L1 was incubated together with ubiquitinated TrkB in deubiquitylation assay buffer at 37°C for 1 h. The ubiquitination of TrkB was analyzed by immunoblotting.