Figure 2.

Cells expressing wild-type and mutated b1-tubulin demonstrate C-terminal polymodification. (A) Constructs carrying wild-type (WT), patient variants (p.R359W and p.L361Afs*19), and a C-terminal tail truncation of b1-tubulin fused to the fluorescent reporter mApple at the N-terminus were designed and cloned. (B) The WT construct was first transfected into Hek293T cells, which were then fixed and immunostained for polyglutamylated and polyglycylated tubulin residues specifically. (C and D) A comparison of the WT and mutated constructs shows a reduction in polyglutamylation and polyglycylation in each mutant compared to the WT. (n=3 independent differentiations, standard deviation plotted on graphs. Two-way ANOVA with multiple comparisons performed to establish significance.)