Fig. 2

Curcumin induced Nrf2 expression by blocking Keap1-Nrf2 interaction. A Normal BEAS-2B cells were treated with (3.125, 6.25, 12.5 μM) curcumin for 24 h. B BEAS-2B cells were treated with 12.5 μM curcumin for 3, 6, 12 and 24 h. Cells were harvested and whole protein lysates were extracted. Nrf2, HO-1, SOD2 and catalase expression were examined by immunoblotting. C BEAS-2B cells were left untreated or were pretreated with 10 μM MG132 for 2 h after which cells were washed and treated with 50 μM CHX either in presence or absence of 12.5 μM curcumin. At different incubation times, cells were lysed and protein levels were evaluated by western blot using Nrf2 specific antibody. Expression of β-actin was evaluated as a loading control. D Curcumin directly bind to the ligand-binding site of Keap1 (PDB code 4L7B). Three-dimensional diagram displays the interaction of curcumin (the yellow stick) and the crystal ligand (the red sticks) to the ligand-binding site of Keap1. Two-dimensional diagram shows the interactions of curcumin to the amino acid residues (e.g., Tyr334, Asn387, Arg415, Ser508. Ser602) in the binding pocket of Keap1. Colors of the residues indicate the forms of interactions with distances as follows: van der Waals forces, green; polarity, magenta. Blue arrows represent H-bonding with the side chain of the amino acid residue. E BEAS-2B cells were co-transfected with expression vectors encoding FLAG-tagged Nrf2 and HA-tagged Keap1. 24 h after transfection cells were then treated with 6.25 or 12.5 μM curcumin along with MG132 (10 μM) for 2 h before cell lysates were collected for ubiquitination assay. Anti-Nrf2 immunoprecipitates were analyzed by immunoblot with anti-ubiquitin antibodies for detection of ubiquitin-conjugated Nrf2. The data are expressed as the mean ± SD of three independent experiments (n = 9). *p < 0.05, **p < 0.01 vs control