Fig. 2

Functional characterization of the identified HINT1 variants. a Western blot analysis of protein extract from HNT1-deleted yeast strain expressing either the wild-type HINT1 or the disease-causing variants. Equal loading was validated with rabbit polyclonal anti-GAPDH antibody and relative HINT1 expression was normalized to hWT. Graph represents relative quantification of band intensities for four independent replicates. b Genetic complementation analysis in HNT1-deleted yeast strain performed by spot assay. Serial dilutions of the different yeast strains were spotted on minimal media without leucine, supplemented with either 2% glucose or 2% galactose, and incubated at 39 °C for 3 days. c Western blot analysis of protein extracts from HINT1-KO HeLa cell lines transiently transfected with either the wild-type HINT1 or the disease-causing variants. Graph represents relative quantification of band intensities for four independent replicates. d Western blot analysis of total protein extracts from HINT1 patients or control (Ctrl) lymphoblasts. Membranes were immunoblotted with polyclonal rabbit anti-human HINT1 antibody. Equal loading was validated with mouse monoclonal anti-β-actin and anti-α-tubulin antibodies and relative HINT1 expression was normalized to hWT and Ctrl expression. Graph represents relative quantification of band intensities for three independent replicates. Statistical one-way ANOVA analysis was performed. Bar charts are presented as means with standard error of the mean (s.e.m.)