Journal: Clinical Cancer Research

Article Title: Morin (3,5,7,2′,4′-Pentahydroxyflavone) Abolishes Nuclear Factor-κB Activation Induced by Various Carcinogens and Inflammatory Stimuli, Leading to Suppression of Nuclear Factor-κB–Regulated Gene Expression and Up-regulation of Apoptosis

doi: 10.1158/1078-0432.ccr-06-2394

Figure Lengend Snippet: Fig. 3. A, effect of morin on theTNF-induced activation of IKK. KBM-5 cells (4 106/mL) were pretreated with 50 Amol/L morin for 4 h and then activated with100 pmol/L TNF for the indicated times.Whole-cell extracts were prepared, and extracts were immunoprecipitated with antibodies against IKK-a and IKK-h.The immune complex kinase assay was then done as described in Materials and Methods.Thereafter, the immunocomplex kinase assay was done as described in Materials and Methods.To examine the effect of morin on the level of expression of IKKa, 50 Ag of cell extract protein were analyzed in 10% SDS-PAGE and assayed for IKKa byWestern blot analysis. B, to detect the direct effect of morin on theTNF-induced activation of IKK, cells were stimulated with100 pmol/LTNF for1h, and then whole-cell extracts were prepared.The extracts were then incubated with different concentrations of morin and immunoprecipitated with anti-IKKa and IKKh antibodies.The immunocomplex kinase assay was then done. C, effect of morin onTNF-induced AKTactivation. Cells (2 106/mL) were incubated with either medium or 50 Amol/L morin for 4 h and then treated with 100 pmol/L TNF for the indicated times.Whole-cell extracts were prepared, and phospho-AKT (Ser473) and AKTwere detected byWestern blot using phospho-specific anti-AKTand anti-AKTantibodies. Results are representative of three independent experiments.

Article Snippet: Anti– IKK-a, anti– IKK-h, and phospho-AKT (Ser473) antibodies were kindly provided by Imgenex (San Diego, CA).

Techniques: Activation Assay, Immunoprecipitation, Immune Complex Kinase Assay, Kinase Assay, Expressing, SDS Page, Incubation