Recombinant glua1

Dimethyl sulfoxide (DMSO) was from Fisher Scientific. AMPA was from Cayman Chemical and NBQX was from Alomone Labs. Recombinant GluA1 and 14-3-3ε were from Origene. Recombinant Nedd4-2 was from Abnova. R18 was from Sigma-Aldrich. Cycloheximide, poly-D-lysine and Protein A/G beads were from Santa Cruz Biotechnology. The antibodies used in this study were purchased from Santa Cruz Biotechnology (anti-α-Tubulin), Cell Signaling (anti-Nedd4-2, anti-pan-14-3-3, anti-N-cadherin and anti-Ubiquitin), Millipore (anti-GluA1), Abcam (anti-MAP2), Thermo Scientific (anti-HA) and GenScript Corporation (anti-Gapdh). The epilepsy-associated mutations were generated using site-directed mutagenesis reagent (Agilent) to introduce mutations into pCI-HA-Nedd4-2 [15 (link)]. The primers used are as below.
S233L: 5’-GGACGTGTCCTCGGAGTTGGACAATAACATCAGAC-3’,
5’-GTCTGATGTTATTGTCCAACTCCGAGGACACGTCC-3’;
E271A: 5’- GGGCGGGGATGTCCCCGCGCCTTGGGAGACCATTTC-3’,
5’- GAAATGGTCTCCCAAGGCGCGGGGACATCCCCGCCC-3’;
H515P: 5’- CGTTTGAAATTTCCAGTACCTATGCGGTCAAAGACATC-3’,
5’- GATGTCTTTGACCGCATAGGTACTGGAAATTTCAAACG-3’.

HA-Ub (Boston Biochem), Ubiquitin Activating Enzyme (UBE1) (Boston Biochem) and UbcH5b/UBE2D2 (Boston Biochem) were obtained. Recombinant WT Nedd4-2 was obtained from Abcam. When HA-tagged WT or mutant Nedd4-2s were produced in HEK cells, 250 μg of total protein lysates were subjected to immunoprecipitation with anti-HA antibody to partially purify HA-tagged Nedd4-2s. Recombinant GluA1 (Origene) was used as substrate for in vitro ubiquitination with recombinant Nedd4-2 (Fig 7A) or Nedd4-2s obtained from transfected HEK cells (Figs 6B and 7C) following a protocol previously described [63 (link)]. Recombinant 14-3-3ε was obtained from Origene.