Figure 3: Parkin ubiquitinates GluK2.
From: Parkin regulates kainate receptors by interacting with the GluK2 subunit
(a) In vitro ubiquitination assay of Myc-GluK2a using recombinant E1 (human ubiquitin-activating enzyme E1), recombinant E2 (UbcH7), HA-ubiquitin and parkin. Myc-GluK2a was slightly ubiquitinated in the presence of E1 and E2 alone but recombinant parkin significantly increased GluK2a ubiquitination. Results are representative of nine independent experiments (two-tailed unpaired t-test, *P=0.0201, t=2.580, df=16). Error bars indicate±s.e.m. (b) Western blotting of co-immunoprecipitation using HEK293T cells transfected with Myc-GluK2a, HA-ubiquitin, wt parkin or parkin C431S. GluK2a was ubiquitinated in HEK293T cells and parkin transfection significantly increased GluK2a ubiquitination. Parkin C431S transfection did not increase GluK2a ubiquitination. Results are representative of three independent experiments (one-way analysis of variance (ANOVA) and Tukey test, *P=0.0219, F=9.029). Error bars indicate±s.e.m. (c) Western blotting of co-immunoprecipitation using primary hippocampal neurons transfected with HA-ubiquitin and infected with lentiviral particle encoding sh-parkin or sh-scrambled, or sh-parkin+parkinR. Endogenous parkin silencing significantly decreased endogenous GluK2 ubiquitination. ParkinR overexpression rescued endogenous GluK2 ubiquitination. The image is representative of three independent experiments that gave identical results (one-way ANOVA and Tukey test, *P=0.0169, F=8.689). Error bars indicate±s.e.m.
