ML120B is a reversible competitive inhibitor of IKKβ and inhibits cytokine production and IκBα phosphorylation and degradation in stimulated bone marrow cells. (A) Molecular structure of ML120B. (B) Inhibition of the IKK complex by ML120B at 50 μM ATP, using biotinylated GST–tagged IκBα (5-55) as a substrate. The data represent the average of triplicate measurements, with error bars indicating SD. (C) ML120B is an ATP competitive inhibitor of the IKK complex. Phosphorylation of biotinylated GST–tagged IκBα (5-55) was measured at fixed inhibitor concentrations of 167 μM (▪), 111 μM (▴), 75 μM (♦), 50 μM (•), 33 μM (□), 22 μM (▵), and 0 μM (⋄). (D) Bone marrow was isolated from a C57BL/6 female mouse and red blood cells were lysed and plated into a 96-well plate (4 × 105 cells/well). Cells were preincubated with ML120B for 1 hour and then stimulated with LPS (1 μg/mL). Supernatants were collected after 4 hours and IL-6 and RANTES levels were evaluated. (E) Western blot analysis of bone marrow preincubated with ML120B (30 μM) or media with equivalent DMSO (0.5%) for 1 hour and then stimulated with LPS (1 μg/mL) for 5, 15, 30, and 60 minutes. (F) Nuclear translocation of p65. Bone marrow cells were stimulated as described in panel D and nuclear extracts were analyzed by immunoblotting. Error bars indicate SD.
