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. Author manuscript; available in PMC: 2022 Mar 5.

Published in final edited form as: Eur J Med Chem. 2021 Jan 2;213:113148. doi: 10.1016/j.ejmech.2020.113148

Figure 4. — PPA inhibits p97 ATPase activity selectively and irreversibly. (A) Titration curves of the in vitro ATPase assays for PPA inhibition of p97, NSF, and Cdc48. (B) PPA did not inhibit human 26S proteasome activity. (C) Reversibility of PPA inhibition was determined using Ub^G76V-GFP by washing out test compound and monitoring decay of GFP signal in the presence of cycloheximide for additional 17 h. (D) PPA block degradation of an ERAD substrate, TCRα-GFP stably expressed in HEK293 cells. Cells were incubated with MG132, washed and treated with cycloheximide plus compound for 60 min or 120 min. Samples were immunoblotted with anti-p97 as loading control and anti-GFP antibody to detect TCRα-GFP.

Figure 4. — PPA inhibits p97 ATPase activity selectively and irreversibly. (A) Titration curves of the in vitro ATPase assays for PPA inhibition of p97, NSF, and Cdc48. (B) PPA did not inhibit human 26S proteasome activity. (C) Reversibility of PPA inhibition was determined using Ub^G76V-GFP by washing out test compound and monitoring decay of GFP signal in the presence of cycloheximide for additional 17 h. (D) PPA block degradation of an ERAD substrate, TCRα-GFP stably expressed in HEK293 cells. Cells were incubated with MG132, washed and treated with cycloheximide plus compound for 60 min or 120 min. Samples were immunoblotted with anti-p97 as loading control and anti-GFP antibody to detect TCRα-GFP.