Epilepsy-associated gene Nedd4-2 mediates neuronal activity and seizure susceptibility through AMPA receptors
(A1) Western blots of Ub or GluA1 after immunoprecipitation with anti-GluA1 antibody following in vitro 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 (A2) and Coomassie blue staining showing the purity of recombinant Nedd4-2 and 14-3-3ε (A3) (n = 4, 2-way ANOVA with post-hoc Tukey test). (B1) 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 (B2) is shown on the right (n = 4, one-way ANOVA with post-hoc Tukey test). (C1) Western blots of Ub or GluA1 after immunoprecipitation with anti-GluA1 antibody following in vitro ubiquitination with recombinant GluA1 in the presence or absence of recombinant 14-3-3ε. HA-tagged WT or mutant Nedd4-2s used for in vitro 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. (C2) 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 t-test. For all experiments, data are represented as mean ± SEM with *p<0.05, **p<0.01.