Fig. 6. NF-L O-GlcNAcylation is dysregulated by CMT-causative mutations.

a NEFL^−/− 293T cells were transfected with WT or CMT mutant NF-L-myc-6xHis + OGT for 24 h and analyzed by myc IP and IB. b Normalized O-GlcNAc signal (O-GlcNAc/myc ratio) was calculated for the experiments performed in (a). Data are shown as mean ± SEM and assessed by one-way ANOVA/Tukey’s post hoc correction (n = 3 biological replicates). The samples from different gels derived from the same experiment, with sample processing controls (NF-L WT) included and gels/blots were processed in parallel. c NEFL^−/− 293T cells were transfected with WT or CMT mutant NF-L-myc-6xHis ± OGT-myc for 24 h and analyzed by differential extraction and IB. d NF-L amount extracted into urea buffer was calculated as percent of total NF-L across three fractions from the experiment described in (c). Data are shown as mean ± SEM and assessed by one-way ANOVA/Tukey’s post hoc correction (n = 3 biological replicates). The samples from different gels derived from the same experiment, with sample processing controls (NF-L WT) included and gels/blots were processed in parallel. e SW13 vim⁻ cells were transfected with WT or CMT mutant NF-L-myc-6xHis + INA-V5 for 24 h and analyzed by IFA. Scale bar: 10 μm. f Quantification of percent of cells with full-length NFs from the experiment described in (e) was performed by a blinded researcher. Data are shown as mean ± SEM and assessed by one-way ANOVA/Tukey’s post hoc correction (n = 3 biological replicates). n.s. not significant. g NEFL^−/− 293T cells were transfected with WT or CMT mutant NF-L-myc-6xHis ± 100 μM Ac₃GlcNDAz-1P(Ac-SATE)₂ for 48 h, subjected to UV cross-linking, and analyzed by IB. Normalized cross-link signal (cross-link/uncross-linked ratio) was calculated. Data are shown as mean ± SEM and assessed by one-way ANOVA/Tukey’s post hoc correction (n = 3 biological replicates). n.s. not significant.