AZTMP inhibits IKKβ-mediated phosphorylation of IκBα.
(A) PEL cells constitutively express IKK. BCBL-1 and BCLM cells were treated with CHX (50 μg/mL) for 1 and 2 hours, and Western blot analysis of whole-cell extracts was performed using antibodies specific for IκBα, with β-actin used as a control. BCLM cells were also treated with CHX for 2 hours in the presence of the proteosome inhibitor LLnL (10 and 20 μM) and assayed for IκBα by Western blot analysis. (B) Phosphorylated AZT (AZTMP) specifically inhibits IKK activity. Top panel: 100 μg cytosolic extract from BCLM cells was immunoprecipitated with anti-IKKα/β polyclonal antibody. The purified enzyme was incubated in vitro with medium, BAY-11 (10 μM), AZT (40 μM), or AZTMP (40, 10, and 4 μM). IKK activity was measured using recombinant GST-IκBα (1-317) as a substrate in the presence of 32P-ATP. Bottom panel: Western blot analysis using the same IKK antibody.
