Figure 3.

EPRS Protein Amount and Activity Are Decreased in Affected Individuals with Hypomyelinating Leukodystrophy-Causing Mutations in EPRS

(A) Immunoblot with protein extracted from hypomyelinating leukodystrophy-affected individuals and age- and sex-matched control fibroblasts. Immunoblot analysis with anti-EPRS revealed a reduced abundance of EPRS, ∼60% of the control amount (control 1 [C1], control 2 [C2]), in individuals P1 and P2. Anti-GAPDH was used as a loading control.

(B) Endogenous amounts of EPRS in fibroblasts between affected individuals and age-sex-matched controls were quantified by a ratio of EPRS band intensity over GAPDH with the three immunoblot analysis experiments, one of which is shown in (A). Abundance of EPRS relative to GAPDH was assessed in three independent experiments using three different protein extracts from each sample. Bar chart shows quantification of EPRS protein amounts compared to control in each condition.

(C) Activity of wild-type and mutated recombinant prolyl-tRNA synthetase protein (p.Pro1115Arg) (nmol released Pi/min/mg protein). Assay was performed at least in triplicate.

(D) 5 μg of total protein was used for determining EPRS activity in lymphoblast lysates from affected individual P4.

Error bars show standard deviation, ^∗p < 0.05.