Fig. 3
From: A case report of late-onset cerebellar ataxia associated with a rare p.R342W TGM6 (SCA35) mutation
In vitro functional analysis. Significant reduction of transamidase activity of TG6-R342W and TG6-R111C in transiently transfected HEK293T cells, showed by immunoblotting analysis (a) and quantification of enzymatic activity (b). The transamidase activity was measured by incubating equal amounts of proteins from the cell lysates with a biotinylated peptide (Biotinyl-Thr-Val-Gln-Gln-Glu-Leu-OH 0.5 mM, Zedira GmbH #B001), which is irreversibly cross-linked to proteins in the presence of DTT (5 mM) and high calcium concentration (5 mM CaCl2), and detected by SDS-PAGE electrophoresis followed by western blotting with an anti-streptavidin antibody conjugated to a fluorophore with emission at 800 nm (LI-COR IRDye 800CW #926–32,230, 1:10000). Images were then acquired with an Odyssey infrared imaging system (LI-COR Biosciences). TG6 activity is shown as fold change compared to TG6-WT activity (set as 1). Graph, mean ± SEM, *P < 0.05, 1-way ANOVA with Tukey’s post hoc test. Original uncropped blots are shown in Additional file 3