Fig. 1. Gossypol acetate bound to VCP and inhibited its ATPase activityin vitro.
  1. 2D-chemical structure of gossypol acetate.
  2. The dose-dependent curve of VCP inhibitory activity for gossypol acetate. IC50 value for inhibition of VCP ATPase activity was measured by the Malachite green-based ATPase assay. The error bar represents Standard Error of Mean (SEM, n = 3).
  3. Partially trypsin digestion analysis suggested direct binding of gossypol acetate to VCP. Purified 6XHis-VCP protein was incubated with DMSO, gossypol, gossypol acetate or NMS873 for 1 h at 37℃ in a total volume of 50μL. After electrophoresis, the gels were stained with Coomassie brilliant blue.
(D) Inhibition on ATPase activities of VCP-FL, VCP-N+D1 or VCP-D1+D2 domain by gossypol acetate as measured by the Malachite green-based ATPase assay. Shown was error (s.d.) of three replicates.**,P<0.01
(E and F) Representative Western-blots and quantifications showed changes in thermodynamic stability of purified 6XHis-VCP in presence of 5μM gossypol acetate. The curves of protein relative degradation were shown with mean and error (SEM) of three replicates.
(G and H) Representative Western-blots and quantifications showed changes in thermodynamic stabilities of purified 6XHis-VCP-N and 6XHis-VCP-N+D1 truncated proteins in presence of 5μM gossypol acetate. The curves of protein relative degradation were shown with error (s.d.) of three replicates.