Pull-down Experiments
Recombinant VCP (residues 1–806) and the N-D1 (residues 1–481),D2(residues 461–806), were incubated with Ace.gos ( 5/10/15uM ) 2hr at 4℃in 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.