Epilepsy-associated gene Nedd4-2 mediates neuronal activity and seizure susceptibility through AMPA receptors
(A1) Western blots of Ubiquitin (Ub) or GluA1 after immunoprecipitation using anti-GluA1 antibody from HEK cells transfected with GluA1 along with HA-tagged WT or mutant Nedd4-2 for 48 hours. Quantification of ubiquitinated GluA1 by the entire area of smear from 100–250 kDa is shown on the right (A2) (n = 4, one-way ANOVA with post-hoc Tukey test). (B1) Western blots of Ub or GluA1 after immunoprecipitation with anti-GluA1 antibody following in vitro ubiquitination with recombinant GluA1. 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. Quantification of ubiquitinated GluA1 by the entire area of smear (B2) and Coomassie blue staining showing the purity of recombinant GluA1 (B3) are shown (n = 4, one-way ANOVA with post-hoc Tukey test). (C1) Western blots of GluA1 and Nedd4-2 after cycloheximide treatment over an 8-hr time course. HEK cells were transfected with GluA1 and HA-tagged WT or mutant Nedd4-2s for 48 hours. Cells were then treated with cycloheximide (100 μg/ml) to inhibit protein translation and follow protein degradation. Representative western blots after 0- and 8-hr cycloheximide treatment (C1) and time courses of GluA1 (C2) and Nedd4-2 (C3) levels after cycloheximide treatment are shown. Analyses were performed by comparing GluA1 or Nedd4-2 level at each time point between cultures receiving different Nedd4-2s (n = 4, one-way ANOVA with post-hoc Tukey test). For all experiments, data are represented as mean ± SEM with *p<0.05, **p<0.01.