Three epilepsy-associated missense mutations of Nedd4-2 disrupt 14-3-3-facilitated GluA1 ubiquitination.

<p>(<b>A</b>_<b>1</b>) Western blots of Ub or GluA1 after immunoprecipitation with anti-GluA1 antibody following <i>in vitro</i> ubiquitination with recombinant GluA1 and Nedd4-2, and in the presence or absence of recombinant 14-3-3ε, 14-3-3 inhibitor R18, or Ubiquitin as labeled. Quantification of lanes 1–4 by the entire area of smear from 100–250 kDa (<b>A</b>_<b>2</b>) and Coomassie blue staining showing the purity of recombinant Nedd4-2 and 14-3-3ε (<b>A</b>_<b>3</b>) (n = 4, 2-way ANOVA with post-hoc Tukey test). (<b>B</b>_<b>1</b>) Western blots of Nedd4-2 or 14-3-3 after a co-immunoprecipitation using anti-14-3-3 antibody with the lysate of HEK cells transfected with HA-tagged WT or mutant Nedd4-2s for 48 hours. Input of transfected Nedd4-2s, endogenous 14-3-3, and Tubulin are shown on the bottom. Quantification of immunoprecipitated Nedd4-2 (<b>B</b>_<b>2</b>) is shown on the right (n = 4, one-way ANOVA with post-hoc Tukey test). (<b>C</b>_<b>1</b>) Western blots of Ub or GluA1 after immunoprecipitation with anti-GluA1 antibody following <i>in vitro</i> ubiquitination with recombinant GluA1 in the presence or absence of recombinant 14-3-3ε. HA-tagged WT or mutant Nedd4-2s used for <i>in vitro</i> ubiquitination were obtained from HEK cells transfected with one of the Nedd4-2s followed by immunoprecipitation with anti-Nedd4-2 antibody. Right before the washing, 1/10 of reaction mixture was obtained and used as input control shown on the bottom. (<b>C</b>_<b>2</b>) The intensity of ubiquitinated GluA1 by the entire area of smear from 100–250 kDa is normalized to the WT group in the absence of 14-3-3ε (lane 3 on the representative blot C₁). The difference in each group with or without the addition of 14-3-3ε was analyzed by Student’s <i>t</i>-test. For all experiments, data are represented as mean ± SEM with *p<0.05, **p<0.01.