Fig. 5.

The IL-1RI is tyrosine-phosphorylated, and the association of p85 subunits to IL-1RI is prevented by the Src inhibitor PP2 but not the MyD88 mimetic AS-1. (A–C) Cultured AH neurons from wild-type mice were developed on a preformed bed of glia from IL-1RI^−/− mice. (D) Cultured AH neurons from MyD88^−/− mice were developed on a preformed bed of glia from IL-1RI^−/− mice. Immunoprecipitations were performed with 750–1,000 μg of total protein. (A) Cells were incubated with 10–12 nM IL-1β for 10 min, and the blot was probed with anti-phosphotyrosine antibody (described in Methods). The representative Western blot is shown from three independent experiments. (B) Wild-type AH cells were incubated with 10–12 nM IL-1β for 10 min after a 15-min exposure to the MyD88 mimetic AS-1 (100 μM) or the Src-family inhibitor PP2 (2 μM). Blots were stripped, and immunoprecipitated proteins were redetected with appropriate antibody, as described in Methods. Relative p85 association was calculated by normalizing p85 staining to IL-1RI staining and comparing relative to control. The representative Western blot is shown, and the bar graph indicates average relative p85 association (±SEM) from two independent experiments. (C) Lower portion of Western blots described above in B were probed with anti-MyD88 antibody (described in Methods). Relative MyD88 association was calculated by normalizing MyD88 staining to IL-1RI staining and comparing relative to control. The representative Western blot is shown, and the bar graph indicates the average relative MyD88 association (±SEM) from two independent experiments. (D) MyD88 ^−/− cells were incubated with 10–12 nM IL-1β for 10 min, and the blot was probed with anti-p85 antibody, stripped, and reprobed with anti-IL-1RI antibody, as described in Methods. The representative Western blot is shown from two independent experiments.