Fig. 3. Gossypol acetate enhanced LC3-VCP-mHTT ternary complex formation by targeting VCP.
(A) In vitro pull-down assays using purified 6XHis-VCP protein incubated with GST-LC3 protein and gossypol acetate. Gossypol acetate or DMSO was added at the indicated concentrations for 1hr before pull-down using GST beads. Input and pull-down samples were detected by Coomassie Brilliant Blue (CBB). Gossypol acetate increased interaction between VCP and LC3 as shown by Western-blots.
(B) In vitro pull-down assays using purified Flag-VCP protein incubated with purified 6XHis-mHTT-Q72 or 6XHis-wtHTT-Q25 protein and gossypol acetate. Gossypol acetate or DMSO was added at the indicated concentrations for 1hr before pull-down using Flag beads. Input and pull-down samples were detected by Western-blot with the indicated antibodies. Gossypol acetate increased interaction between VCP and mHTT-Q72, but had little influence on VCP’s interaction with wtHTT-Q25.
(C) In vitro pull-down assays using purified 6XHis-VCP protein, GST-LC3 proteins and 6XHis-mHTT-Q72 or 6XHis-wtHTT-Q25 protein in the absence or presence of gossypol acetate. Gossypol acetate or DMSO was added at the indicated concentrations for 1hr before pull-down using GST beads. Input and pull-down samples were detected by Western-blot with the indicated antibodies. Gossypol acetate enhanced formation of LC3-VCP-mHTT complex.
(D) Enhanced LC3-VCP interaction by gossypol acetate depended on the presence of both N and D1 domain. In vitro pull-down assays were performed using purified 6XHis-VCP protein, VCP N domain, N+D1 or D1+D2 truncated proteins. Gossypol acetate or DMSO was added at the indicated concentrations for 1hr before pull-down using GST-beads. Input and pull-down samples were detected by Coomassie brilliant blue (CBB).