Supplementary Figure 7: α-synuclein(ΔDQ) and α-synuclein A53T are degraded by TAT-βsynCTM.

The endogenous α-synuclein CMA targeting motif was eliminated by mutating ⁹⁵VKKDQ⁹⁹ into ⁹⁵VKKAA⁹⁹. Plasmids expressing wild-type α-synuclein, α-synuclein (ΔDQ) and α-synuclein A53T were transfected into HEK293 cells and treated with either TAT-βsyn (50μM) or TAT-βsynCTM (50μM) starting at 24h post-transfection. Two additional doses of peptide were added 4 and 8h later, and cells were harvested at 48h after transfection. Wild-type α-synuclein was significantly degraded by TAT-βsynCTM, which was rescued by inhibiting the lysosome with NH₄Cl (n=4, H(2)=8.290, p=0.005, Kruskal-Wallis One-way ANOVA on Ranks). α-synuclein(ΔDQ) was degraded by TAT-βsynCTM (n=4, F(2,11)=9.482, p=0.006, One-way ANOVA), which was rescued by NH₄Cl (n=4, p=0.001). Similarily, α-synuclein A53T was degraded by the targeting peptide (n=4, F(2,11)=6.340, p=0.019, One-way ANOVA), which was rescued by NH₄Cl (n=4, p=0.019). Student-Newman-Keuls was used for post-hoc analysis. β-actin was used as loading control. * compared to TAT-βsyn, Δ compared to TAT- βsynCTM *^,Δ p<0.05, **p<0.01; bars represent relative mean values±s.e.m. Cells were collected from at least 4 separate cultures and transfections. Full-length blots are available in Supplementary Figure 9.