Fig. 6

The pathogenicity of SPAST-Y52C mutation identified in a patient with HSP. A Structure of SPAST-M1 protein and its domains with the location of the mutations previously identified from patients with HSP. B The tyrosine 52 residue to cysteine mutant of SPAST (Y52C), identified from a Japanese patient with HSP, was constructed using primers to include the desired change. Interaction between SPAST WT or Y52C and FBXL17 in HEK293 cells transfected with indicated plasmids. C The poly-ubiquitination of WT and Y52C of Flag-SPAST-M1 from nucleus fraction of HEK293 cells transfected with indicated plasmids. D and E HeLa cells were transfected with Flag-SPAST-M1 WT or Y52C and treated with 100 μM H₂O₂ for 24 h. And then, cells were stained with JC-1 fluorescence dye and quantified with ImageJ software. (Scale bar: 50 μm). F Protein expressions of ER stress and apoptosis-related genes in HeLa cells transfected with WT or Y52C of Flag-SPAST-M1 in presence or absence of H2O2 treatment. G HeLa cells treated as in (D) was counted with an automated cell counter. All experiments were performed in duplicate, data are expressed as the mean of two samples with SD, and results are representative of two independent experiments. (*P < 0.05; ** P < 0.01; ns, not significant)