Fig. 5

Parkin-mediated GluN1 ubiquitination is impaired in pathogenic mutants. a Representative immunoblots for GFP immunoprecipitation (IP) from HEK293T cell lysates expressing Myc/Myc-Parkin, GFP-GluA1/-GluA2/-GluN1/-GluN2B, and HA-ubiquitin, probed for HA and GFP. Ubiquitin immunoreactivity used for quantification is marked on HA blots. b Quantification of GFP-GluA or GluN ubiquitination, expressed as the ratio of marked HA blot intensity (a) with Myc-Parkin (+) to Myc control (−), then normalized to immunoprecipitated GFP, GFP-GluA or GluN (n = 3 experiments, *P < 0.05; one-way ANOVA, error bars represent SEM). c Representative Myc and GFP immunoblots for Myc IP from HEK293T cell lysates expressing Myc-Parkin and GFP-GluA1/-GluA2/-GluN1/-GluN2B. Arrowhead indicates immunoprecipitated Myc-Parkin (just below IgG band). d Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/C431S/W403A, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. e Quantification of Flag-GluN1 ubiquitination by measurement of marked HA blot intensity (d), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP control (n = 3 experiments, **P < 0.01, ***P < 0.001, one-way ANOVA, error bars represent SEM). f Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/T240M/R275W/R334C/G430D constructs, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. g Quantification of Flag-GluN1 ubiquitination by measurement of marked HA intensity (f), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP condition (n = 3 experiments, *P < 0.05; **P < 0.01, ***P < 0.001, one-way ANOVA, error bars represent SEM)