FIGURE 4.

NEFL expression in allelic series of edited motor neurons. A series of edited iPSCs derived from a healthy control (WT) and CMT2E patient were differentiated into i³LMNs, and RNA was extracted on day 7 or protein on day 10. (A) Total NEFL transcript expression by quantitative RT-ddPCR, normalized to GAPDH. (B) Total NF-L protein expression by western blot, normalized to GAPDH. (C) Relative expression of NEFL alleles in i³LMNs derived from CMT2E patient line and edited subclones. Allele-specific RT-ddPCR targeting a heterozygous SNP in the 3′ UTR was used to genotype transcripts using linkage shown in the schematic. All bar graphs represent mean ± S.E.M. For all graphs ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Šídák’s test for multiple comparisons.