Fig. 5

UBE2K binds directly to GluN2B and facilitates its ubiquitination. a Protein levels of GluN2B and UBE2K in N2a cells overexpressing UBE2K were examined by WB (n = 3). b Protein levels of GluN2B and UBE2K in N2a cells transfected with UBE2K shRNA (shUBE2K) or control (shControl) for 72 h (n = 3). c Immunoprecipitation was performed using 500 μg of proteins from cell lysates transfected with vector or UBE2K-GFP fused plasmid, using nonspecific IgG (IgG) and anti-GFP antibodies. The resulting precipitates were blotted with antibodies against GluN2B, RNF168, RNF138, RNF2, or UBE2K. Additionally, 30 μg of protein from the extracts without immunoprecipitation were loaded as input. d Immunoprecipitation was performed using 500 μg of proteins extracted from the hippocampus of 4-month-old C57BL/6 mice, using IgG and anti-GluN2B antibodies. The resulting precipitates were blotted with antibodies against GluN2B and UBE2K. e Cultured mouse cortical neurons at DIV14 were stained with antibodies against GluN2B (green) and UBE2K (red), as well as DAPI (blue). Scale bar, 20 μm. f An ubiquitination assay was performed to analyze GluN2B ubiquitination in the hippocampus of 4-month-old WT and APP/PS1 mice, as well as in N2a cells transfected with UBE2K for 48 h. g WB was performed to assess the effect of shUBE2K on the protein degradation rate of GluN2B in the presence of CHX (n = 3). h Using GPS-Uber (http://gpsuber.biocuckoo.cn/online.php), five ubiquitination sites (K2, K25, K1082, K1097, and K1293) on GluN2B were predicted and highlighted on the peptide sequence. Subsequently, we generated three fragments of GluN2B, each containing one of these ubiquitination sites. i N2a cells were co-transfected with UBE2K-GFP and WT or K1082R/K1097R GluN2B-2 fragment for 48 h. Proteins were pulled down using IgG and anti-Flag antibodies, and further detected by WB using antibodies against GluN2B-2 (Flag) and UBE2K (GFP). Data are presented as mean ± SEM and two-tailed t tests were used unless otherwise specified. *P < 0.05, **P < 0.01