Pull-down Experiments
Recombinant VCP (residues 1β806) and the N-D1 (residues
1β481),D2(residues 461β806), were incubated with Ace.gos ( 5/10/15uM )
2hr at 4βin 0.6ml buffer(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2%
Triton-X-100) followed by the addition of GST/GST-LC3/Htt-Q25/Htt-Q72
for 2h. Then incubation with GST-beads for 1 h. Immunoprecipitates were
washed four times with 1XPBS,0.2% Triton-X-100 buffer and were analysed
by SDS-PAGE and Western-blot analysis.