Figure 5

A. S100A4 induces phosphorylation of IKKα/β. Western blot of immunoprecipitated IKK complex from II-11b cells treated with 2 μM S100A4 for the indicated time periods and analyzed for expression of phosphorylated IKKα/β. IKKα was used as loading control. B. H-7 and staurosporine inhibit IKK-mediated IκBα phosphorylation in vitro. Upper panel: Kinase assay showing phosphorylation of recombinant IκBα by immunoprecipitated IKK complex from unstimulated II-11b cells or cells stimulated with 2 μM S100A4 for 15 minutes, with or without H-7 (30 μM) or staurosporine (0.4 μM) present in the reaction mixture. Lower panel: Western blot showing IKKα as a loading control. C. Densitometric quantification of the signals shown in Fig. 5B. The bars show IκBα phosphorylation relative to IKKα expression. One of three independent experiments is quantified due to high background in the other experiments. D. Treatment with H-7 or staurosporine did not influence the level of phosphorylated IKKα/β upon S100A4-stimulation for 15 minutes. Immunoprecipitated IKK complex was analyzed by immunoblotting using anti-phospho IKKα/β antibody. IKKα is used as loading control. E. Densitometric quantification of Fig. 5D. Bars show phosphorylated IKKα/β relative to IKKα expression. B and D are representative results of three independent experiments. St = staurosporine.