CDDO directly interacts with purified Lon and forms a complex with Lon in cultured cells. (A) CDDO^biotin (2.5 μM) was reacted with purified Lon (2 μγ) in PBS containing 1% NP40 for 90 minutes at room temperature, after which CDDO^biotin was pulled down with SA-sepharose (SA-seph). (B) Granta cells were treated with or without CDDO or CDDO^biotin for 1 hour at 37°C, followed by extraction of cellular proteins and incubation with SA-seph as indicated for 90 minutes at 4°C; (C-D) LS174T or Granta cells extracts (20 μg), respectively, were reacted with or without CDDO or CDDO^biotin for 90 minutes at 4°C, followed by SA-seph as in panel B. Pull-down reactions were immunoblotted with antibodies to Lon, ClpP, or BiP. Untreated mock samples contained input but without CDDO^biotin and SA-seph.