Figure 4.

Mutant huntingtin (mHtt) binds cystathionine γ-lyase (CSE) and inhibits its catalytic activity. (A) Interaction of CSE with huntingtin. HEK293 cells were transfected with constructs encoding myc-tagged CSE and either HD17 or HD75 for 24 h and immunoprecipitation conducted using anti-myc antibody. CSE bound both wild-type and mutant Htt. (B) Expressions of CSE, WTHtt, and mHtt at 48 and 96 h post-transfection as determined by Western blotting. (C,D) Activity of CSE at 48 and 96 h, respectively, as measured by assaying H₂S production from 10 mM L-cysteine in the presence of the cofactor, pyridoxal 5-phosphate (PLP at 250 mM), using the methylene blue assay. (E,F) Enzymatic activity of CSE at 48 and 96 h respectively, as measured by assaying cysteine production from cystathionine in the presence of the cofactor, pyridoxal 5-phosphate (PLP at 250 mM), using the ninhydrin assay. n = 3, SEM, *** p < 0.001.