Fig. 3

Functional analysis of novel mutations in SPAST gene. a Schematic of the human M1- and M87-spastin expression vectors for missense mutation c.1040A > C (p.Q347P). The weak Kozak sequence tgaATGa is present at the M1 initiation codon. A better Kozak sequence ctcATGg is present at the M87 initiation codon. b Time-course stability analysis of mutant spastin (M1-p.Q347P) by western blot . Cells were collected at 0, 3, 6, 9, 12 h following treatment with CHX. c Statistical analysis of b (n = 3). All values are normalized to untreated controls. d RT-PCR analysis of mutation c.1245 + 1_c.1245 + 2 insT. Two bands (size in 375 bp and 303 bp) were observed in the missplicing mutation. e Schematic diagram showing the skipping of exon 9 for c.1245 + 1_c.1245 + 2 insT mutation (red arrow indicated). f Sanger sequencing confirmed that the skipping of exon 9 had occurred. Error bars indicate SEM. *, P < 0.05. ns, not significant (P > 0.05)